Multivalent recombinant modified vaccinia virus ankara (MVA) vector encoding filovirus immunogens

ABSTRACT

The present invention relates to an improved filovirus vaccine comprising a recombinant modified vaccinia virus Ankara-based (MVA-based) vaccine against filovirus infection and to related products, methods and uses. Specifically, the present invention relates to genetically engineered (recombinant) MVA and FPV vectors comprising at least one heterologous nucleotide sequence encoding an antigenic determinant of a Marburg virus (MARV) or Ebola virus glycoprotein. Specifically, the invention relates to recombinant MVA comprising Ebola virus glycoprotein and virion protein 40. The invention also relates to products, methods and uses thereof as well as prime/boost regimens of MVA and genetically engineered (recombinant) FPV, e.g., suitable to induce a protective immune response in a subject.

FIELD OF THE INVENTION

The present invention relates to an improved filovirus vaccinecomprising a recombinant modified vaccinia virus Ankara-based(MVA-based) vaccine against filovirus disease and to related products,methods and uses. Specifically, the present invention relates togenetically engineered (recombinant) MVA vectors comprising aheterologous nucleotide sequence encoding an antigenic determinant of afilovirus protein. The present invention also relates to vaccinationmethods, in particular homologous and heterologous prime-boostvaccination regimes employing two viral vector compositions. Moreparticularly, the invention relates to a recombinant MVA for use in ahomologous prime-boost vaccination regime and/or a recombinant MVA and arecombinant fowlpox virus (FPV) for use in a heterologous prime-boostvaccination regime. The invention also relates to products, methods anduses thereof, e.g., suitable to induce a protective immune response in asubject.

BACKGROUND OF THE INVENTION

Filoviruses are enveloped, non-segmented, negative-strand RNA viruses ofthe virus family Filoviridae. Two members of this virus family have beenidentified to date: Marburg virus (MARV) and Ebola virus (EBOV).Filoviruses are extremely virulent, easily transmissible fromperson-to-person, and extraordinarily lethal, causing severe hemorrhagicfever in humans and non-human primates. Filovirus infections have afatality rate in humans ranging from 23% to as high as 90%. Despitetheir transmissibility and lethality, however, no approved therapy orpreventive vaccine is available.

During outbreaks, isolation of patients and use of protective clothingand disinfection procedures (together called viral hemorrhagic fever(VHF) isolation precautions or barrier nursing) has been sufficient tointerrupt further transmission of Marburg or Ebola viruses, and thus tocontrol and end the outbreak. Because there is no known effectivetreatment for the hemorrhagic fevers caused by filoviruses, transmissionprevention through application of VHF isolation precautions is currentlythe only available means to control filovirus outbreaks.

The first filovirus was recognized in 1967 after a number of laboratoryworkers in Germany and Yugoslavia, who had been handling tissues fromAfrican green monkeys, developed severe hemorrhagic fever. A total of 31cases and seven deaths were associated with these outbreaks. The viruswas named Marburg virus (MARV) after Marburg, Germany, the site of oneof the outbreaks. After the initial outbreaks the virus disappeared anddid not reemerge until 1975, when a traveler, most likely exposed inZimbabwe, became ill in Johannesburg, South Africa; the traveler'straveling companion and a nurse were also infected. A few sporadic casesof Marburg hemorrhagic fever (MHF) have been identified since that time,but the disease remains relatively rare.

The second filovirus, Ebola virus (EBOV), was first identified in 1976when two outbreaks of Ebola hemorrhagic fever (EHF) occurred in northernZaire (now the Democratic Republic of Congo) and southern Sudan. Theoutbreaks involved viruses which eventually proved to be two differentspecies of Ebola virus, which were named after the nations in which theywere discovered. Both viruses proved to be highly lethal, with 90% ofthe cases in Zaire and 50% of the cases in Sudan resulting in death.Since 1976, Ebola virus has appeared sporadically in Africa, with a fewsmall-to medium-sized outbreaks confirmed between 1976 and 1979, andagain in Gabon between 1994 and 1996. Larger epidemics of Ebola HFoccurred in Kikwit, Zaire in 1995 and in Gulu, Uganda in 2000.

It appears that filoviruses are transmitted to humans from ongoing lifecycles in one or more non-human animals. Despite numerous attempts tolocate the natural reservoir or reservoirs of Ebola and Marburg viruses,however, their origins remain mysterious. Consequently, it also remainsunclear just how the virus is transmitted from its natural reservoir(s)to humans. Once a human has been infected, however, further infectionsoccur by person-to-person transmission. Specifically, transmissioninvolves close personal contact between an infected individual or theirbody fluids and another person. During recorded outbreaks of hemorrhagicfever caused by filovirus infection, people who cared for (i.e., fed,washed, medicated) or worked very closely with infected individuals wereespecially at risk of becoming infected themselves. Nosocomial(hospital) transmission through contact with infected body fluids (i.e.,via reuse of unsterilized syringes, needles, or other medical equipmentcontaminated with these fluids) has also been an important factor in thespread of disease. Minimizing close contact between uninfected andinfected patients usually reduces the number of new filovirus infectionsin humans during an outbreak. Although filoviruses have displayed somecapability of infection through small-particle aerosols in thelaboratory, airborne spread among humans has not been clearlydemonstrated.

Five strains of Ebola virus have been identified so far, and are namedafter their site of first appearance: Bundibugyo (BEBOV), Ivory Coast(EBOV-Cdl, also called Tai Forest virus or TAFV), Reston (EBOV-Reston),Sudan (SEBOV), and Zaire (ZEBOV); the Zaire, Sudan, and Bundibugyostrains are commonly involved in morbidity and death in humans.Ebola-Reston is the only known filovirus that does not cause severedisease in humans, although it can be fatal in monkeys. Several strainsof Marburg virus have been identified so far, with the Musoke strainhaving the highest lethality rate. See FIG. 1.

Structurally, filovirus virions may appear in several shapes, includinglong, sometimes branched filaments, as well as shorter filaments shapedlike a “6”, the letter “U”, or a circle. Viral filaments can measure upto 14 micrometers (μm) in length, have a uniform diameter of 80nanometers (nm), and are enveloped in a lipid membrane. Each virioncontains one single-stranded, negative-sense RNA molecule approximately19 kilobase pairs (kb) in length, which contains seven sequentiallyarranged genes in the order of nucleoprotein (NP), virion protein 35(VP35), virion protein 40 (VP40), envelope glycoprotein (GP), virionprotein 30 (VP30), virion protein 24 (VP24), and RNA-directed RNApolymerase protein (L). Upon entry into the host cell cytoplasm, the RNAis transcribed to generate polyadenylated, subgenomic mRNA speciesencoding the proteins. Transcription and translation lead to thesynthesis of seven structural polypeptides, with presumed identicalfunctions for each of the different filoviruses. Four proteins (NP,VP30, VP35 and L) are associated with the viral genomic RNA in thenucleocapsid complex. The three remaining structural proteins aremembrane-associated; GP is a type I transmembrane protein, while VP24and VP40 are probably located on the inner side of the membrane. Theenvelope glycoprotein (GP) appears in the viral envelope as a homotrimer(also referred to as a ‘peplomer’) comprising three copies of aheterodimer. The heterodimer contains two fragments of the full-lengthGP precursor (referred to as ‘GP0’) known as ‘GP1’ and ‘GP2’ produced byfurin cleavage. GP1 and GP2 are linked by a disulfide bond. Anon-structural, secreted glycoprotein (sGP) is expressed by EBOV, butnot MARV (H. Feldmann & M. P. Kiley, Curr. Top. Microbiol. Immunol.235:1-21 (1999)). New viral particles are created by budding from thesurface of host cells (see below).

The filovirus life cycle begins with virion attachment to specificcell-surface receptors, followed by fusion of the virion envelope withcellular membranes and release of the virus nucleocapsid into thecytosol. The viral RNA-directed RNA polymerase (RNAP, also known as the‘L’ protein) partially uncoats the nucleocapsid and transcribes thegenes into positive-stranded mRNAs, which are then translated intostructural and nonstructural proteins. See FIG. 2. The RNAP binds to asingle promoter located at the 3′ end of the genome. Transcriptioneither terminates after a gene or continues to the next gene downstream,meaning that genes close to the 3′ end of the genome are transcribed inthe greatest abundance, while those towards the 5′ end of the genome areleast likely to be transcribed. Gene order is therefore a simple buteffective form of transcriptional regulation. The most abundant proteinproduced is the nucleoprotein (NP), cellular concentration of whichdetermines when the RNAP switches from gene transcription to genomereplication. Replication results in full-length, positive-strandedanti-genomes that are in turn transcribed into negative-stranded virusprogeny genome copies. Newly synthesized structural proteins and genomesself-assemble and accumulate near the inside of the cell membrane. Virusparticles are enveloped as they bud from the infected host cell,producing mature infectious virions.

Prior Vaccine Development

Many strategies have been evaluated during attempts to develop a safe,immunogenic vaccine capable of inducing protective immunity againstinfection by one or more filovirus species, with decidedly mixedresults. An overview is summarized in Marzi and Feldmann (A. Marzi andH. Feldmann Expert Rev. Vaccines 13(4):521-531 (2014)). For instance,while a trivalent DNA vaccine comprising a mixture of three DNAplasmids, one expressing the envelope glycoprotein from ZEBOV, a secondexpressing the envelope glycoprotein from SEBOV, and a third expressingthe nucleoprotein from ZEBOV was safe, immunogenic, and able to inducean antibody response against at least one of the three antigens inhumans. CD8+ T-cell responses were detected in fewer than ⅓ of thevaccinated population (J. E. Martin et al., Clin. Vaccine Immunol.13(11):1267-1277 (2006)). Similarly, a complex, pentavalentadenovirus-based ‘pan-filovirus’ vaccine comprising a mixture of fourdifferent recombinant adenoviruses expressing envelope glycoproteinsfrom ZEBOV, SEBOV, Marburg-Ci67 (strain Ratayczak), Marburg-Musoke, andMarburg-Ravn, as well as nucleoproteins from ZEBOV and Marburg-Musoke,protected non-human primates from ZEBOV or MARV challenge and inducedantibody responses to both types of virus, although it remains unclearwhether the vaccine induced any CD8+ T-cell response (D. L. Swenson etal., Clin. Vaccine Immunol. 15(3):460-467 (2008)).

Intranasal administration of a recombinant paramyxovirus—humanparainfluenza virus, serotype 3 (HPIV3)—expressing either the envelopeglycoprotein or both the envelope glycoprotein and nucleoprotein fromZEBOV protected guinea pigs from subsequent challenge with EBOV. Rodentmodels are frequently poorly predictive of results in primates, with anumber of previous EBOV vaccine candidates that were effective inrodents failing completely in non-human primates (A. Bukreyev et al., J.Virol. 80(5):2267-2279 (2006)). Intranasal administration of arecombinant HPIV3 expressing either the envelope glycoprotein or boththe envelope glycoprotein and nucleoprotein from ZEBOV in rhesus monkeysshowed that any construct expressing the envelope glycoprotein wasmoderately immunogenic and protected more than 80% of the animalsagainst disease after post-vaccination challenge with ZEBOV (A. Bukreyevet al., J. Virol. 81(12):6379-6388 (2007)). Finally, a recombinantvesicular stomatitis virus (VSV) in which the VSV glycoprotein wasreplaced by the ZEBOV envelope glycoprotein protected 50% of guineapigs, 100% of mice following treatment as late as 24 hours after anotherwise uniformly lethal infection. Four out of eight rhesus macaques(50%) were protected when treated 20 to 30 min after exposure providinga post-exposure treatment option for Ebola virus infection (H. Feldmann,PLoS Pathogens 3(1):54-61 (2007)).

Geisbert et al. evaluated the effects of vaccine strategies that hadprotected mice or guinea pigs from lethal EBOV infection in nonhumanprimates. They used RNA replicon particles derived from an attenuatedstrain of Venezuelan equine virus (VEEV) expressing EBOV glycoproteinand nucleoprotein, recombinant Vaccinia virus (VACV) expressing EBOVglycoprotein, liposomes containing lipid A and inactivated EBOV, and aconcentrated, inactivated whole-virion preparation. They found that noneof these strategies successfully protected nonhuman primates from robustchallenge with EBOV (T. H Geisbert et al., Emerging Infectious Diseases8(3):503-507 (2002)).

Others have used Virus Like Particles (VLPs) expressed in mammalian,bacterial, plant or insect cells as non-replicating subunit vaccines (D.L. Swenson et al., Vaccine 23:3033-3042 (2005); K. L. Warfield et al.,JID 196(2):430-437 (2007), N. Kushnir et al., Vaccine 31(1):58-83(2012), K. L. Warfield ET AL., PLOS ONE 10(3):e0118881 (2015), K. L.Warfield and M. J. Aman JID 204:1053-1059 (2011), V. M. Wahl-Jensen etal., J Virol. 79(16):10442-10450 (2005), WO 2003/039477, WO 2006/046963,WO 2006/073422, WO 2004/042001, U.S. Pat. Nos. 8,900,595, 7,211,378) toinduce antibody responses. However, filovirus VLPs require acost-intensive and challenging production process and need to be storedat ambient temperature over time.

Thus, after expending considerable time and effort, a few promisingvaccine candidates have emerged at preclinical stages, but at present noapproved preventive vaccine is available. Given the transmissibility andlethality of filovirus infection, there is a pressing need for aneffective vaccine.

BRIEF SUMMARY OF THE INVENTION

It is discovered in the present invention that various prime-boostcombinations of replication deficient and replication incompetentvectors generate effective immune protection against filovirusinfection.

Accordingly, one general aspect of the present invention relates to acombination vaccine comprising:

-   -   a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of at least one filovirus subtype, together        with a pharmaceutically acceptable carrier; and    -   b) a second composition comprising an immunologically effective        amount of a fowlpox vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

In an additional aspect, the present invention relates to a combinationvaccine comprising:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

In an additional aspect, the present invention relates to a kitcomprising:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of at least one filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a fowlpox vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

In an additional aspect, the present invention relates to a kitcomprising:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

In an additional aspect, the present invention relates to a recombinantModified Vaccinia Virus (MVA) vector comprising a nucleotide sequenceencoding two or more antigenic determinants of a filovirus protein foruse in the treatment and/or prevention of a filovirus-caused disease. Inyet another aspect, the invention relates to a recombinant MVA vectorcomprising a nucleotide sequence encoding an antigenic protein of afilovirus glycoprotein and encoding a filovirus virion protein 40 (VP40)for use in the treatment and/or prevention of a filovirus-causeddisease. In another embodiment, the invention relates to a recombinantMVA vector comprising a nucleotide sequence selected from the groupconsisting of a) SEQ ID NO:5, SEQ ID NO:19 and SEQ ID NO:30, b) SEQ IDNO:5, SEQ ID NO:19, SEQ ID NO:28 and SEQ ID NO:30 and c) SEQ ID NO:19and SEQ ID NO:33. In a certain aspect, the invention relates to acomposition comprising said recombinant MVA vector, a vaccine comprisingsaid recombinant MVA vector, a pharmaceutical comprising saidrecombinant MVA vector and a pharmaceutical carrier, diluent and/oradditive, and a cell comprising said recombinant MVA vector. In acertain aspect, the invention relates to said recombinant MVA vector foruse as a medicament or vaccine for treating and/or preventing afilovirus-caused disease in a subject and a method for affecting animmune response in a subject comprising administering to the subjectsaid recombinant MVA vector. In an additional aspect, the presentinvention relates to a kit comprising said recombinant MVA vector in afirst vial or container for a first administration (priming) and in asecond vial or container for a second administration (boosting).

The present invention also relates to a recombinant FPV vectorcomprising a nucleotide sequence encoding at least one antigenicdeterminant of a filovirus protein (e.g. any of the filovirus proteinsas mentioned supra or infra, preferably an filovirus envelopeglycoprotein) under the control of the FPV-40K promoter having SEQ IDNO:26. In an additional aspect, the invention relates to a recombinantfowlpox virus (FPV) vector comprising a nucleotide sequence encodingone, two or more antigenic determinants of a filovirus protein for usein the treatment and/or prevention of a filovirus-caused disease. In acertain aspect, the invention relates to a composition comprising saidrecombinant FPV vector, a vaccine comprising said recombinant FPVvector, a pharmaceutical comprising said recombinant FPV vector and apharmaceutical carrier, diluent and/or additive and a cell comprisingsaid recombinant FPV vector. In a certain aspect, the invention relatesto said recombinant FPV vector for use as a medicament or vaccine fortreating and/or preventing a filovirus-caused disease in a subject and amethod for affecting an immune response in a subject comprisingadministering to the subject said recombinant FPV vector.

In an additional aspect, the present invention relates to a combinationvaccine comprising:

-   -   (a) an immunologically effective amount of a MVA vector        comprising a nucleic acid encoding antigenic proteins of at        least two filovirus subtypes, together with a pharmaceutically        acceptable carrier; and    -   (b) an immunologically effective amount of a fowlpox vector        comprising a nucleic acid encoding an antigenic protein of a        first filovirus subtype, together with a pharmaceutically        acceptable carrier;        -   wherein one of the vectors is a priming vaccine and the            other vector is a boosting vaccine.

In an additional aspect, the present invention relates to a combinationvaccine comprising:

-   -   (a) an immunologically effective amount of a MVA vector        comprising a nucleic acid encoding antigenic proteins of at        least two filovirus subtypes, together with a pharmaceutically        acceptable carrier; and    -   (b) an immunologically effective amount of one or more        additional MVA vectors comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the MVA vectors is a priming vaccine and the            other MVA vectors is a boosting vaccine.

In an additional aspect, the present invention relates to a combinationvaccine comprising:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein;    -   OR    -   (c) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein; and    -   (d) a second composition comprising an immunologically effective        amount of an FPV vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein;    -   wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

In an additional aspect, the present invention relates to a method ofinducing an immune response against a filovirus in a subject, the methodcomprising administering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of at least one filovirus subtype, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a fowlpox vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

In an additional aspect, the present invention relates to a method ofinducing an immune response against a filovirus in a subject, the methodcomprising administering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

The invention also covers a method of generating a recombinant MVAvector for use in the treatment and/or prevention of a filovirus-causeddisease comprising the steps of:

-   -   (a) infecting a host cell with a MVA virus,    -   (b) transfecting the infected cell with a recombinant vector        comprising at least one nucleotide sequence encoding an        antigenic determinant of any of the filovirus proteins of any of        the embodiments of the invention, said nucleic acid sequence        further comprising a genomic MVA virus sequence capable of        directing the integration of the at least one nucleotide        sequence into the MVA virus genome, and    -   (c) identifying, isolating and optionally purifying the        generated recombinant MVA virus.

In another embodiment, the order of step a) and b) of the method ofgenerating a recombinant MVA vector of any of the above embodiments canbe changed such that step b) is the first step and a) the second.

The invention also covers a method of generating a recombinant FPVvector for use in the treatment and/or prevention of a filovirus-causeddisease comprising the steps of:

-   -   (a) infecting a host cell with an FPV virus,    -   (b) transfecting the infected cell with a recombinant vector        comprising at least one nucleotide sequence encoding an        antigenic determinant of any of the filovirus proteins of any of        the embodiments of the invention, said nucleic acid sequence        further comprising a genomic FPV virus sequence capable of        directing the integration of the at least one nucleotide        sequence into the FPV virus genome, and    -   (c) identifying, isolating and optionally purifying the        generated recombinant FPV virus.

In another embodiment, the order of step a) and b) of the method ofgenerating a recombinant FPV vector of any of the above embodiments canbe changed such that step b) is the first step and a) the second.

In an additional aspect, the present invention relates to a method ofinducing an immune response against a filovirus in a subject comprisingadministering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein;    -   OR    -   (c) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein; and    -   (d) a second composition comprising an immunologically effective        amount of an FPV vector comprising a nucleic acid encoding at        least one antigenic determinant of a filovirus protein    -   wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

In an additional aspect, the present invention relates to a method ofproviding protective immunity or a protective immune response in asubject, the method comprising administering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of at least one filovirus subtype, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a fowlpox vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

In an additional aspect, the present invention relates to a method ofproviding protective immunity or a protective immune response in asubject comprising administering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        -   wherein one of the compositions is a priming composition and            the other composition is a boosting composition.

In an additional aspect, the present inventions relates to a method forproduction of filovirus-like particles in a subject comprisingadministering to the subject:

-   -   (a) an immunologically effective amount of a MVA vector        comprising a nucleic acid encoding antigenic proteins of at        least one filovirus glycoprotein and a filovirus virion protein        40 (VP40), together with a pharmaceutically acceptable carrier;        and    -   (b) an immunologically effective amount of a fowlpox vector or a        MVA vector comprising a nucleic acid encoding an antigenic        protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;    -   wherein one of the vectors is a priming vaccine and the other        vector is a boosting vaccine.

In an additional aspect, the present inventions relates to a method forproduction of filovirus-like particles in a subject comprisingadministering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least one filovirus glycoprotein and a        filovirus virion protein 40 (VP40), together with a        pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a fowlpox vector or a MVA vector comprising a nucleic        acid encoding an antigenic protein of a first filovirus subtype,        together with a pharmaceutically acceptable carrier;    -   wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

In an additional aspect, the invention relates to a method of inducingan enhanced immune response against a filovirus in a subject, the methodcomprising production of filovirus-like particles in the subject byadministering to the subject:

-   -   (a) an immunologically effective amount of a MVA vector        comprising a nucleic acid encoding antigenic proteins of at        least one filovirus glycoprotein and a filovirus virion protein        40 (VP40), together with a pharmaceutically acceptable carrier;        and    -   (b) an immunologically effective amount of a fowlpox vector or a        MVA vector comprising a nucleic acid encoding an antigenic        protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;    -   wherein one of the vectors is a priming vaccine and the other        vector is a boosting vaccine.

In an additional aspect, the invention relates to a method of inducingan enhanced immune response against a filovirus in a subject, the methodcomprising production of filovirus-like particles in the subject byadministering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least one filovirus glycoprotein and a        filovirus virion protein 40 (VP40), together with a        pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a fowlpox vector or a MVA vector comprising a nucleic        acid encoding an antigenic protein of a first filovirus subtype,        together with a pharmaceutically acceptable carrier;    -   wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

FIG. 1 shows a phylogenetic tree depicting the relationships betweenvarious identified filovirus strains. The tree was constructed usingcoding regions of envelope glycoprotein (GP) genes and the maximumparsimony method. Both the Ravn and Ratayczak strains of Marburg virushad a 23% fatality rate, while the Musoke and Angola strains hadfatality rates ranging from 50% to 88%. The Sudan strain had a 41-65%fatality rate, and the Zaire strain had a 57-90% fatality rate. Both theCote d'Ivoire and Reston strains have not yet caused disease in man,though Reston has caused disease in pigs.

FIG. 2 shows the structure and genetic organization of the filovirusgenome.

FIG. 3A shows the structure and genetic organization of MVA-mBN252B.FIG. 3B shows the structure and genetic organization of MVA-mBN226B.FIG. 3C shows the structure and genetic organization of MVA-mBN254Aincluding the selection marker. FIG. 3D shows the structure and geneticorganization of MVA-mBN368A including the selection marker.

FIG. 4A shows the structure and genetic organization of plasmid pBNX186.Flank 1 (F1 IGR 88/89) and flank 2 (F2 IGR 88/89) are sequences ofMVA-BN surrounding IGR 88/89. F1 IGR 88/89 and F2 IGR 88/89 are used forinsertion of the expression cassette and the selection cassette (NPT IIand eGFP) into MVA-BN in a homologous recombination event. The E. colidrug selection gene Neomycin Phosphotransferase (NPT II) and an enhancedGreen Fluorescent Protein (eGFP) were connected via an internalribosomal entry site (IRES) and inserted under the control of a strongsynthetic poxvirus promoter (PrS) in order to allow selection forrecombinant viruses. F2 and F2-repeat sequences of IGR 88/89 flank theselection cassette enabling the removal of the selection cassette viahomologous recombination in the absence of selective pressure.

FIG. 4B shows the structure and genetic organization of plasmid pBNX197.Flank 1 (F1 IGR 148/149) and flank 2 (F2 IGR 148/149) are sequences ofMVA-BN surrounding IGR 148/149. F1 IGR 148/149 and F2 IGR 148/149 areused for insertion of the expression cassette and the selection cassette(GPT and RFP) into MVA-BN in a homologous recombination event. The E.coli Guanine-Xanthine-Phosphoribosyl-Transferase drug selection gene(GPT) and a Red Fluorescence Protein gene (RFP) were inserted as afusion gene under the control of a strong synthetic poxvirus promoter(PrS) in order to allow selection for recombinant viruses. LoxPsequences flank the selection cassette enabling the Crerecombinase-mediated removal of the selection cassette. FIG. 4C showsthe structure and genetic organization of plasmid pBN274, whichexpresses Cre recombinase. FIG. 4D shows the structure and geneticorganization of plasmid pBNX221. Flank 1 (F1 IGR BamHI J FowlPox) andflank 2 (F2 IGR BamHIJ FowlPox) are sequences of FPV surrounding theinsertion site BamHI J. F1 IGR BamHI J FowlPox and F2 IGR BamHI JFowlPox are used for insertion of the expression cassette and theselection cassette (GPT and RFP) into FPV in a homologous recombinationevent. The E. coli Guanine-Xanthine-Phosphoribosyl-Transferase drugselection gene (GPT) and a Red Fluorescence Protein gene (RFP) wereinserted as a fusion gene under the control of a strong syntheticpoxvirus promoter (PrS) in order to allow selection for recombinantviruses. LoxP sequences flank the selection cassette enabling the Crerecombinase-mediated removal of the selection cassette. FIG. 4E showsthe structure and genetic organization of plasmid pBNX214. Flank 1 (F1IGR 148/149) and flank 2 (F2 IGR 148/149) are sequences of MVA-BNsurrounding IGR 148/149. F1 IGR 148/149 and F2 IGR 148/149 are used forinsertion of the expression cassette and the selection cassette (GPT andRFP) into MVA-BN in a homologous recombination event. pBNX214 alreadyincludes the PrS5E promoter for the expression of transgenes. The E.coli Guanine-Xanthine-Phosphoribosyl-Transferase drug selection gene(GPT) and a Red Fluorescence Protein gene (RFP) were inserted as afusion gene under the control of a strong synthetic poxvirus promoter(PrS) in order to allow selection for recombinant viruses. LoxPsequences flank the selection cassette enabling the Crerecombinase-mediated removal of the selection cassette.

FIG. 5A shows the structure and genetic organization of plasmid pBN433.The GP-MARV-Musoke was inserted under control of the promoter PrS intothe BspEI/NheI site of pBNX197. In addition the plasmid also containsMVA-BN DNA sequences flanking the IGR 148/149 of the MVA-BN genome andthe loxP-flanked selection cassette. The loxP sites allow the laterelimination of the selection cassette by Cre recombinase-mediatedrecombination. FIG. 5B shows the structure and genetic organization ofplasmid pBN384. The glycoprotein genes of Ebola virus Zaire-Mayinga(GP-ZEBOV-Mayinga) and Marburg virus Musoke (GP-MARV-Musoke) wereinserted under control of the promoters Pr7.5 and PrS into the MluI/NheIsites of pBNX197. In addition, the plasmid also contains MVA-BN DNAsequences flanking the IGR 148/149 of the MVA-BN genome and theloxP-flanked selection cassette. The loxP sites allow the laterelimination of the selection cassette by CRE recombinase-mediatedrecombination. FIG. 5C shows the structure and genetic organization ofplasmid pBN385. The glycoprotein gene of Ebola virus Sudan (GP-SEBOV)and the Nucleoprotein of Ebola virus Ivory Coast (NP-EBOV-Cdl) wereinserted under control of the synthetic promoters PrS and PrLE1 into theMluI/NheI sites of pBNX186. In addition, the plasmid also containsMVA-BN DNA sequences flanking the IGR 148/149 of the MVA-BN genome and aselection cassette flanked by F2 and F2rpt in order to allow the laterelimination of the selection cassette via homologous recombination inthe absence of selective pressure. FIG. 5D shows the structure andgenetic organization of plasmid pBN436. The glycoprotein gene of Ebolavirus Zaire-Mayinga (GP-ZEBOV-Mayinga) was inserted into the BspEI/NotIsites of pBNX214 under control of the PrS5E promoter. In addition, theplasmid also contains MVA-BN DNA sequences flanking the IGR 148/149 ofthe MVA-BN genome and the loxP-flanked selection cassette. The loxPsites allow the later elimination of the selection cassette by Crerecombinase-mediated recombination. FIG. 5E shows the structure andgenetic organization of plasmid pBN555. The glycoprotein gene of Ebolavirus Zaire-Mayinga (GP-ZEBOV-Mayinga) under control of the FPV-40Kpromoter was inserted into the MluI/NotI sites of pBNX221. In addition,the plasmid also contains FPV DNA sequences flanking the Insertion siteBamHI J of the FPV genome and the loxP-flanked selection cassette. TheloxP sites allow the later elimination of the selection cassette by Crerecombinase-mediated recombination.

FIG. 6 shows the levels of antibodies against GP in cynomolgus macaquesfollowing vaccination with MVA-BN-Filo (MVA-mBN226B) as measured byELISA. Animals were vaccinated twice four weeks apart with MVA-BN-Filo(on Day −42 and Day −14), and blood was drawn at intervals for analysisvia ELISA: prior to vaccination (Day −42, red curve (1)), after thefirst but prior to the second vaccination (Day −14, green curve (2)),and after the second vaccination (Day −5, orange curve (3)). The graphon the left shows Marburg GP specific antibodies in serum, the graph inthe middle shows Ebola Zaire GP specific antibodies in serum, and thegraph on the right shows Ebola Sudan GP specific antibodies in serum.Hyperimmune serum from cynomolgus macaques immunized with either MarburgAngola GP (left graph), Ebola Zaire GP (middle graph), or Ebola Sudan GP(right graph) was used as positive control in each ELISA.

FIG. 7A and FIG. 7B show the results of vaccination with MVA-BN-Filo(MVA-mBN226B) following challenge with MARV-Musoke. FIG. 7A shows thatvaccination with MVA-BN-Filo protected 100% of animals from challengewith MARV-Musoke. FIG. 7B shows clinical scores post-challenge;vaccinated animals challenged with MARV-Musoke showed no symptoms orhistological changes associated with hemorrhagic fever and harbored novirus in liver, spleen, adrenal glands, lymph nodes, or lungs.

FIG. 8 shows the antibody and CD8 T cell response after heterologousMVA/FPV immunization. H-2K^(k+) B6CBA F1 mice were immunized s.c. with5×10⁷ TCID₅₀ MVA-ZEBOV-GP (MVA; MVA-mBN354A, see FIG. 3C) orFPV-ZEBOV-GP (FPV; FPVmBN368A, see FIG. 3D) on day 0 and 21. A) Micewere bled on day 21 and 41 for antibody analysis. Shown is the meanconcentration of ZEBOV-GP-specific antibodies +/− SEM. B) On day 41,mice were sacrificed and spleens were analyzed flow-cytometrically afterre-stimulation with GP₅₇₇₋₅₈₄ peptide. Shown is the absolute number ofCD107a⁺, IFN-γ⁺ and TNF-α⁺ CD8 T cells per spleen×10⁴+/− SEM.rMVA=recombinant MVA-ZEBOV-GP (MVA-mBN254); rFPV=recombinantFPV-ZEBOV-GP (FPV-mBN368).

FIG. 9 shows the ZEBOV-GP specific CD8 T cell response afterimmunization (s.c) of mice with MVA/FPV. Shown is the absolute number ofCD107a⁺, IFN-γ⁺ and TNF-α⁺ CD8 T cells per spleen x 10⁴+/− SEM. 1:MVA-mBN254/FPV-mBN368; 2: MVA-mBN226/FPV-mBN368, 3:MVA-mBN255/FPV-mBN368.

FIG. 10 shows ZEBOV-GP specific antibodies of cynomolgus macaques whichreceived prime-boost vaccinations on Study Day 0 and 28 withMVA-BN-ZEBOV/GP (MVA-mBN254) at a dose of 5×10⁸ TCID₅₀ (n=3), withMVA-BN-ZEBOV/GP-VP40 (MVA-mBN255) at a dose of 5×10⁸ TCID₅₀ (n=3)according to Example 6. Results are presented as the geometric meanconcentration (ng/ml) together with the standard error of the mean(SEM).

FIG. 11 shows neutralizing antibody responses of cynomolgus macaqueswhich received three vaccinations on Study Day 0, 28 and withMVA-BN-ZEBOV/GP at a dose of 5×10⁸ TCID₅₀ (n=2), or withMVA-BN-ZEBOV/GP-VP40 (5×10⁸ TCID₅₀, n=2). Additional animals (n=2)received TBS as negative control on Study Day 0 and 56. Sera wereanalyzed by ZEBOV-GP-specific pseudo virion neutralizing assay. Resultsare presented as individual antibody titer neutralizing 80% of ZEBOV-GPexpressing VSV.

FIGS. 12 A) and B) shows the formation of filovirus-like particles inHeLa cells infected with MVA-BN-ZEBOV/GP-VP40 (MVA-mBN255). A, B)Transmission electron microscopy (TEM) analysis of MVA-BN-ZEBOV/GP-VP40(VLP) and MVA wt infected HeLa cells. HeLa cells were infected withMVA-BN-ZEBOV/GP-VP40 (A) or BAC-derived MVA-wt (B) at an MOI of 10 andthin sections were generated and processed for TEM. Arrow: Transversesection of VLP generated by MVA-BN-ZEBOV/GP-VP40. C) Shows an immunoblotanalysis of (co-)expression of GP and VP40 in Hela cells. D) Shows animmunoblot of immunoprecipitates from the supernatants of HeLa cells(aliquots of the same supernatants as shown in C) infected withMVA-BN-ZEBOV/GP-VP40 at an MOI of 10 for 2 days. VP40 and GP can only beco-precipitated if present in intact VLPs but not after disruption ofVLPs with Triton-X-100 (1%). 166: MVA-mBN166, 254: MVA-mBN254, 255:MVA-mBN255.

FIG. 13 shows the structure of certain recombinant MVA/FPV constructs.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that a vaccine comprising a recombinantmodified vaccinia virus Ankara (MVA) comprising a heterologousnucleotide sequence encoding an antigenic determinant of a Marburg virus(MARV) glycoprotein (GP) provides a filoviral vaccine capable ofinducing both cellular and humoral responses sufficient to conferprotective immunity to Marburg virus, as well as to smallpox. Theinsertion of additional heterologous nucleotide sequences encoding anantigenic determinant of an Ebola virus Zaire (ZEBOV) glycoprotein (GP),Ebola Virus Sudan (SEBOV) glycoprotein (GP), and/or an EBOVnucleoprotein (NP) into the recombinant MVA produces a multivalentvaccine capable of inducing immune responses to both MARV and EBOV, andeven to multiple strains of MARV and/or EBOV, such as, for example SudanEbola virus (SEBOV) and Zaire Ebola virus (ZEBOV), the two typesassociated with the lethal forms of Ebola hemorrhagic fever. Thus, arecombinant MVA vector comprising a nucleotide sequence encoding anantigenic determinant of an EBOV GP reveals very good immune responsesagainst Ebola strains. Moreover, the excellent safety profile of MVA andits derivatives (e.g., MVA-BN), as well as their ability to accommodatemultiple heterologous nucleotide sequences enables the production of asafe single component multivalent pan-filovirus vaccine, in contrast toa number of multi-component vaccines in early stages of development (seebelow).

Given the fact that prior art attempts to generate an immune responseagainst filoviruses, in particular in non-human primates against MARVand EBOV, failed, the present invention came as a surprise. It could nothave been expected from what is taught and what was achieved in theprior art that a MVA-based vaccine would generate an immune responsethat confers protection in non-human primates against filovirusinfection, in particular against MARV. Of course, from the datagenerated by the present inventors and their observations, it is morethan reasonable and plausible to conclude that the MVA-based vaccinewould also induce an immune response in humans. Indeed, the FDA acceptsnon-human primate models as proof that a vaccine which confersprotection in these non-human primates is likewise suitable in humans.

The present inventors have also found that a vaccination regimecomprising a recombinant modified vaccinia virus Ankara (MVA) comprisinga heterologous nucleotide sequence encoding an antigenic determinant ofEBOV, such as, for example Sudan Ebola virus (SEBOV) and/or Zaire Ebolavirus (ZEBOV) in combination with a recombinant modified FPV comprisinga heterologous nucleotide sequence encoding an antigenic determinant ofEBOV, for example Sudan Ebola virus (SEBOV) and/or Zaire Ebola virus(ZEBOV) provides a filoviral vaccine capable of inducing both cellularand humoral responses sufficient to confer protective immunity.

In the study underlying the present invention it has also been foundthat the use of a MVA vector comprising a nucleic acid encoding anantigenic protein of at least one filovirus subtype, in particular afilovirus glycoprotein, and a fowlpox vector comprising at least onenucleic acid encoding an antigenic protein of a first filovirusglycoprotein as a heterologous prime and boost generates a protectiveimmune response against a filovirus immunogen by induction of a highlevel of antibody response and an up to 5-fold higher cytotoxic CD8 Tcell response, in particular wherein the MVA vector was used as at leastone prime composition and the fowlpox as a boost composition.

The recombinant MVA and/or FPV may be either monovalent, i.e.,comprising only one heterologous sequence encoding an antigenicdeterminant of EBOV, or multivalent, i.e., comprising at least twoheterologous sequences encoding antigenic determinants of EBOV.

The invention thus provides vaccines or vaccine combinations for use ingenerating an immune response that confers dual protection or crossprotection against infections by at least two filovirus subtypes inparticular Marburg virus and/or Ebola virus subtypes and vaccines orvaccine combinations which can be used for manufacturing of a vaccineagainst at least two filovirus subtypes in particular Marburg virusand/or Ebola virus subtypes. Thus, vaccines for cross-protection againstfiloviruses such as Ebola Zaire-Mayinga and Zaire-Kikwit and/orMarburg-Musoke and Marburg-Angola could be provided. It is now alsodiscovered for the first time, that immunization with a MVA vectorexpressing certain antigens such as the VP40 protein of ZEBOV togetherwith other heterologous nucleotide sequences encoding for at least onesurface glycoprotein of a filovirus, in particular of ZEBOV, cangenerate filovirus-like particles e.g., Ebola virus-like particlescontaining the filovirus glycoprotein on their surface. This wasunexpected since it had been reported that transport of filoviral GP tothe cell surface was largely inhibited by MVA (Sanger et al. J. Virol.Meth. 81, 29-35 (2001)). However, since filovirus particle buddingoccurs at the cell surface (Noda et al., PLoS Pathog. 2(9):e99 (2006))efficient GP surface transport is required for formation ofGP-containing filovirus-VLP. In the study underlying the presentinvention the recombinant MVA expressing filovirus virion protein 40(VP40) and a glycoprotein e.g., GP-ZEBOV-Mayinga capable of producingVLPs induced an enhanced immune response with various prime-boostcombinations and protected non-human primates against filovirusinfection. The studies performed could also show that a homologousprime-boost based solely on recombinant MVA expressing a filovirusglycoprotein and a filovirus virion protein 40 (VP40) protein protectedagainst a filovirus infection in non-human primates.

It has further been found that the use of a MVA vector comprising anucleic acid encoding an antigenic glycoprotein of at least onefilovirus subtype, in particular a glycoprotein of a Marburg virusand/or Ebola virus, and a nucleic acid encoding an antigenic protein ofa virion protein 40 (VP40) as a heterologous prime boost with a fowlpoxvector comprising at least one nucleic acid encoding an antigenicprotein of a first filovirus glycoprotein generates an enhanced CD8 Tcell response. In was further found that the use of a MVA vectorcomprising a nucleic acid encoding an antigenic glycoprotein of at leastone filovirus subtype, in particular a glycoprotein of an Ebola virusand a nucleic acid encoding an antigenic protein of a virion protein 40(VP40) induced a higher neutralizing antibody response in non-humanprimates e.g., already after priming which was further improved afterboosting and thus generates an immune response against one or morefilovirus infections, in particular Zaire-Mayinga and Zaire-Kikwit. Ithas also been shown that immunization with a MVA vector expressingcertain antigens such as the filovirus virion protein 40 (VP40) togetherwith a filovirus glycoprotein can produce VPLs that express a filovirusenvelope glycoprotein lining the entire surface of the VLPs whichresemble intact filovirus virions. In this way, incorporation of anucleic acid encoding for a filovirus VP40 protein into the MVA vectorwas shown to enhance the immune response of the viral vector expressingthe antigenic protein or proteins, in particular the MVA vector.

Reference will now be made in detail to exemplary embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings.

Recombinant MVA Virus

In one aspect, the present invention provides a recombinant modifiedvaccinia virus Ankara (MVA) comprising a nucleotide sequence encoding anantigenic determinant of a filovirus glycoprotein (GP), in particular anenvelope glycoprotein. In another aspect, the present invention providesa recombinant MVA vector comprising a heterologous nucleotide sequenceencoding an antigenic determinant of a filovirus glycoprotein, inparticular an envelope glycoprotein, and a heterologous nucleotidesequence encoding an antigenic determinant of a further filovirusprotein. MVA has been generated by more than 570 serial passages onchicken embryo fibroblasts of the dermal vaccinia strain Ankara(Chorioallantois vaccinia virus Ankara virus, CVA; for review see Mayret al. (1975), Infection 3: 6-14) that was maintained in the VaccinationInstitute, Ankara, Turkey for many years and used as the basis forvaccination of humans. However, due to the often severe post-vaccinationcomplications associated with vaccinia viruses, there were severalattempts to generate a more attenuated, safer smallpox vaccine.

During the period of 1960 to 1974, Prof. Anton Mayr succeeded inattenuating CVA by over 570 continuous passages in CEF cells (Mayr etal. (1975)). It was shown in a variety of animal models that theresulting MVA was avirulent (Mayr, A. & Danner, K. (1978), Dev. Biol.Stand. 41:225-234). As part of the early development of MVA as apre-smallpox vaccine, there were clinical trials using MVA-517 incombination with Lister Elstree (Stickl (1974), Prev. Med. 3:97-101;Stickl and Hochstein-Mintzel (1971), Munch. Med. Wochenschr.113:1149-1153) in subjects at risk for adverse reactions from vaccinia.In 1976, MVA derived from MVA-571 seed stock (corresponding to the571^(st) passage) was registered in Germany as the primer vaccine in atwo-stage parenteral smallpox vaccination program. Subsequently, MVA-572was used in approximately 120,000 Caucasian individuals, the majoritychildren between 1 and 3 years of age, with no reported severe sideeffects, even though many of the subjects were among the population withhigh risk of complications associated with vaccinia (Mayr et al. (1978),Zentralbl. Bacteriol. (B) 167:375-390). MVA-572 was deposited at theEuropean Collection of Animal Cell Cultures as ECACC V94012707.

As a result of the passaging used to attenuate MVA, there are a numberof different strains or isolates, depending on the number of passagesconducted in CEF cells. For example, MVA-572 was used in a small dose asa pre-vaccine in Germany during the smallpox eradication program, andMVA-575 was extensively used as a veterinary vaccine. MVA as well asMVA-BN lacks approximately 13% (26.6 kb from six regions) of the genomecompared with ancestral CVA virus. The deletions affect a number ofvirulence and host range genes, as well as the gene for Type A inclusionbodies. MVA-575 was deposited on Dec. 7, 2000, at the EuropeanCollection of Animal Cell Cultures (ECACC) under Accession No.V00120707. The attenuated CVA-virus MVA (Modified Vaccinia Virus Ankara)was obtained by serial propagation (more than 570 passages) of the CVAon primary chicken embryo fibroblasts.

Even though Mayr et al. demonstrated during the 1970s that MVA is highlyattenuated and avirulent in humans and mammals, certain investigatorshave reported that MVA is not fully attenuated in mammalian and humancell lines since residual replication might occur in these cells(Blanchard et al. (1998), J. Gen. Virol. 79:1159-1167; Carroll & Moss(1997), Virology 238:198-211; U.S. Pat. No. 5,185,146; Ambrosini et al.(1999), J. Neurosci. Res. 55: 569). It is assumed that the resultsreported in these publications have been obtained with various knownstrains of MVA, since the viruses used essentially differ in theirproperties, particularly in their growth behaviour in various celllines. Such residual replication is undesirable for various reasons,including safety concerns in connection with use in humans.

Strains of MVA having enhanced safety profiles for the development ofsafer products, such as vaccines or pharmaceuticals, have been developedby Bavarian Nordic: MVA was further passaged by Bavarian Nordic and isdesignated MVA-BN. A representative and preferred sample of MVA-BN wasdeposited on Aug. 30, 2000 at the European Collection of Cell Cultures(ECACC) under Accession No. V00083008. MVA-BN is further described in WO02/42480 (US 2003/0206926) and WO 03/048184 (US 2006/0159699), both ofwhich are incorporated by reference herein.

MVA-BN can attach to and enter human cells where virally-encoded genesare expressed very efficiently. MVA-BN is strongly adapted to primarychicken embryo fibroblast (CEF) cells and does not replicate in humancells. In human cells, viral genes are expressed, and no infectiousvirus is produced. MVA-BN is classified as Biosafety Level 1 organismaccording to the Centers for Disease Control and Prevention in theUnited States. Preparations of MVA-BN and derivatives have beenadministered to many types of animals, and to more than 2000 humansubjects, including immune-deficient individuals. All vaccinations haveproven to be generally safe and well tolerated. Despite its highattenuation and reduced virulence, in preclinical studies MVA-BN hasbeen shown to elicit both humoral and cellular immune responses tovaccinia and to heterologous gene products encoded by genes cloned intothe MVA genome (E. Harrer et al. (2005), Antivir. Ther. 10(2):285-300;A. Cosma et al. (2003), Vaccine 22(1):21-9; M. Di Nicola et al. (2003),Hum. Gene Ther. 14(14):1347-1360; M. Di Nicola et al. (2004), Clin.Cancer Res., 10(16):5381-5390).

“Derivatives” or “variants” of MVA refer to viruses exhibitingessentially the same replication characteristics as MVA as describedherein, but exhibiting differences in one or more parts of theirgenomes. MVA-BN as well as a derivative or variant of MVA-BN fails toreproductively replicate in vivo in humans and mice, even in severelyimmune suppressed mice. More specifically, MVA-BN or a derivative orvariant of MVA-BN has preferably also the capability of reproductivereplication in chicken embryo fibroblasts (CEF), but no capability ofreproductive replication in the human keratinocyte cell line HaCaT(Boukamp et al (1988), J. Cell Biol. 106:761-771), the human boneosteosarcoma cell line 143B (ECACC Deposit No. 91112502), the humanembryo kidney cell line 293 (ECACC Deposit No. 85120602), and the humancervix adenocarcinoma cell line HeLa (ATCC Deposit No. CCL-2).Additionally, a derivative or variant of MVA-BN has a virusamplification ratio at least two fold less, more preferably three-foldless than MVA-575 in Hela cells and HaCaT cell lines. Tests and assayfor these properties of MVA variants are described in WO 02/42480 (US2003/0206926) and WO 03/048184 (US 2006/0159699).

The term “not capable of reproductive replication” or “no capability ofreproductive replication” is, for example, described in WO 02/42480,which also teaches how to obtain MVA having the desired properties asmentioned above. The term applies to a virus that has a virusamplification ratio at 4 days after infection of less than 1 using theassays described in WO 02/42480 or in U.S. Pat. No. 6,761,893.

The term “fails to reproductively replicate” refers to a virus that hasa virus amplification ratio at 4 days after infection of less than 1.Assays described in WO 02/42480 or in U.S. Pat. No. 6,761,893 areapplicable for the determination of the virus amplification ratio.

The amplification or replication of a virus is normally expressed as theratio of virus produced from an infected cell (output) to the amountoriginally used to infect the cell in the first place (input) referredto as the “amplification ratio”. An amplification ratio of “1” definesan amplification status where the amount of virus produced from theinfected cells is the same as the amount initially used to infect thecells, meaning that the infected cells are permissive for virusinfection and reproduction. In contrast, an amplification ratio of lessthan 1, i.e., a decrease in output compared to the input level,indicates a lack of reproductive replication and therefore attenuationof the virus.

The advantages of MVA-based vaccine include their safety profile as wellas availability for large scale vaccine production. Preclinical testshave revealed that MVA-BN demonstrates superior attenuation and efficacycompared to other MVA strains (WO 02/42480). An additional property ofMVA-BN strains is the ability to induce substantially the same level ofimmunity in vaccinia virus prime/vaccinia virus boost regimes whencompared to DNA-prime/vaccinia virus boost regimes.

The recombinant MVA-BN viruses, the most preferred embodiment herein,are considered to be safe because of their distinct replicationdeficiency in mammalian cells and their well-established avirulence.Furthermore, in addition to its efficacy, the feasibility of industrialscale manufacturing can be beneficial. Additionally, MVA-based vaccinescan deliver multiple heterologous antigens and allow for simultaneousinduction of humoral and cellular immunity.

In a preferred embodiment, the recombinant MVA vector of any of theembodiments used for generating the recombinant virus is a MVA-BN virusor a derivative having the capability of reproductive replication invitro in chicken embryo fibroblasts (CEF) cells, but no capability ofreproductive replication in the human keratinocyte cell line HaCat, thehuman bone osteosarcoma cell line 143B, the human embryo kidney cellline 293, and the human cervix adenocarcinoma cell line HeLa.

In another embodiment, the recombinant MVA vector of any of theembodiments used for generating the recombinant virus is MVA-BN asdeposited at the European Collection of Animal Cell cultures (ECACC)under accession number V00083008.

MVA vectors useful for the present invention can be prepared usingmethods known in the art, such as those described in WO 02/042480 and WO02/24224, both of which are incorporated by reference herein.

In another aspect, a MVA viral strain suitable for generating therecombinant virus may be strain MVA-572, MVA-575 or any similarlyattenuated MVA strain. Also suitable may be a mutant MVA, such as thedeleted chorioallantois vaccinia virus Ankara (dCVA). A dCVA comprisesdel I, del II, del III, del IV, del V, and del VI deletion sites of theMVA genome. The sites are particularly useful for the insertion ofmultiple heterologous sequences. The dCVA can reproductively replicate(with an amplification ratio of greater than 10) in a human cell line(such as human 293, 143B, and MRC-5 cell lines), which then enable theoptimization by further mutation useful for a virus-based vaccinationstrategy (see WO 2011/092029).

Recombinant FPV

In one aspect, the present invention provides a recombinant FPVcomprising a nucleotide sequence encoding an antigenic determinant of afilovirus glycoprotein (GP), in particular an envelope glycoprotein. Inanother aspect, the present invention provides a recombinant FPVcomprising a heterologous nucleotide sequence encoding an antigenicdeterminant of a filovirus glycoprotein, in particular an envelopeglycoprotein, and a heterologous nucleotide sequence encoding anantigenic determinant of a further filovirus protein.

An FPV according to the invention is a prototype species within thegenus of the Avipoxvirus. Numerous FPV strains are described and areavailable for example from CEVA Laboratories, Cynamid Webster, FortDodge, Intercontinental Laboratories, Intervet (NOBILIS VARIOLE), Merial(DIFTOSEC CT strain), Schering-Plough, Select Laboratories, Solvay,Syntro-Zeon and Vineland Laboratories. FP1 is a Duvette strain modifiedto be used as a vaccine in one day old chickens. The strain is acommercial fowlpox virus vaccine strain designated 0 DCEP 25/CEP67/2309October, 1980 and is available from Institute Merieux, Inc. FP5 is acommercial fowlpox virus vaccine strain of chicken embryo originavailable from American Scientific Laboratories (Division of ScheringCorp.) Madison, Wis., United States Veterinary License No. 165, serialNo. 30321. Various attenuated strains of fowlpox virus are known such asFPV M (mild vaccine strain) and FPV S (standard vaccine strain)obtainable from Cyanamid Websters PtY, Ltd Australia. The US Departmentof Agriculture (USDA) challenge strain has been further described by C.L. Afonso et al., J. Virol. 74(8):3815-3831 (2000), 74(8):3815-3831(2000). FP9 is a fowlpox strain used for vaccine purposes obtained inthe late 1980s by Tomeley, Binns, Boursnell and Brown at the IAHHoughton Laboratories (St Ives, UK). It was derived from plaquepurification of a virus that had been passaged 438 times in chickenembryo fibroblasts (CEF) culture from HP1 (A. Mayr & K. Malicki (1966),Zentralbl Veterinarmed (B) 13:1-13, Skinner et al. (2005), Expert Res.Vaccines 4(1):63-76). Other attenuated strains are PDXVAC-TC as suchdescribed in S. Jenkins et al. (1991), Aids Research and HumanRetroviruses 7(12):991:998. Deposited strains encompass for examplefowlpox virus ATCC® VR-229 (typical fowlpox scabs from combs of chickensin New Jersey prior to 1928) and fowlpox virus ATCC® VR-250 (chicken,Kentucky, 1950).

In another aspect, a FPV viral strain suitable for generating therecombinant virus can be any strain mentioned supra or any similar FPVstrain. In another aspect, the FPV is selected from the group of FP1,FP5, FP9, FPV M, FPV S, ATCC® VR-229, ATCC® VR-250, the USDA strain andPDXVAC-TC. In yet another embodiment, the FPV of any of the embodimentsis an attenuated FPV.

An advantage of FPV is that the virus causes disease only in avianspecies, but is able to enter and express transgenes in mammalian cells,while being immunologically non-cross-reactive with vaccinia virus andcan thus escape pre-existing immunity in smallpox-experienced humans.

Recombinant FPV vectors suitable for generating the recombinant FPV canbe constructed by well-established methods. Live attenuated fowlpoxviruses may be produced by multiple passage of the virus in avian cells.The preparation of the FPV vectors is described, for example in MichaelJ. P. Lawman and Patricia D. Lawman (eds.) Cancer Vaccines: Method andProtocols, Methods in Molecular Biology, vol. 1139, Chapter 32 Paul M.Howley, Kerrilyn R. Diener and John D. Hayball p. 407-427. Thegeneration of recombinant FPV useful for virus-based vaccinationstrategy has also been described in EP 0 284 416 B1, WO 88/02022, WO89/03429, WO 89/03879, WO89/07644, WO 89/12684, WO 90/02191, WO91/02072, WO 89/03879 and WO 94/019014. The genome sequence and genomeorganization has been described by Afonso et al. and Laidlaw and Skinner(C. L. Afonso et al. (2000), J. Virol. 74(8):3815-3831, S. M. Laidlawand M. A. Skinner (2004), Journal of General Virology 85:305-322). Anexemplary genome sequence of FPV can be found in GenBank Accession No.AF198100.1.

Antigenic Determinants

The term “antigenic determinant” refers to any molecule that stimulatesa host's immune system to make an antigen-specific immune response,whether a cellular response or a humoral antibody response. Antigenicdeterminants may include proteins, polypeptides, antigenic proteinfragments, antigens, and epitopes which still elicit an immune responsein a host and form part of an antigen, homologues or variants ofproteins, polypeptides, and antigenic protein fragments, antigens andepitopes including, for example, glycosylated proteins, polypeptides,antigenic protein fragments, antigens and epitopes, and nucleotidesequences encoding such molecules. Thus, proteins, polypeptides,antigenic protein fragments, antigens and epitopes are not limited toparticular native nucleotide or amino acid sequences but encompasssequences identical to the native sequence as well as modifications tothe native sequence, such as deletions, additions, insertions andsubstitutions.

The term “epitope” refers to a site on an antigen to which B- and/orT-cells respond, either alone or in conjunction with another proteinsuch as, for example, a major histocompatibility complex (“MHC”) proteinor a T-cell receptor. Epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by secondary and/ortertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents, whileepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 5, 6, 7, 8,9, 10 or more amino acids—but generally less than 20 amino acids—in aunique spatial conformation. Methods of determining spatial conformationof epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., “Epitope MappingProtocols” in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed(1996).

Preferably, a homologue or variant has at least about 50%, at leastabout 60% or 65%, at least about 70% or 75%, at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically, at leastabout 90%, 91%, 92%, 93%, or 94% and even more typically at least about95%, 96%, 97%, 98% or 99%, most typically, at least about 99% identitywith the referenced protein, polypeptide, antigenic protein fragment,antigen and epitope at the level of nucleotide or amino acid sequence.

Techniques for determining sequence identity between nucleic acids andamino acids are known in the art. Two or more sequences can be comparedby determining their “percent identity.” The percent identity of twosequences, whether nucleic acid or amino acid sequences, is the numberof exact matches between two aligned sequences divided by the length ofthe shorter sequences and multiplied by 100.

“Percent (%) amino acid sequence identity” with respect to proteins,polypeptides, antigenic protein fragments, antigens and epitopesdescribed herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe reference sequence (i.e., the protein, polypeptide, antigenicprotein fragment, antigen or epitope from which it is derived), afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forexample, using publically available computer software such as BLAST,ALIGN, or Megalign (DNASTAR) software. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximum alignment over the full-length ofthe sequences being compared.

The same applies to “percent (%) nucleotide sequence identity”, mutatismutandis.

For example, an appropriate alignment for nucleic acid sequences isprovided by the local homology algorithm of Smith and Waterman, (1981),Advances in Applied Mathematics 2:482-489. This algorithm can be appliedto amino acid sequences by using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res.14(6):6745-6763. An exemplary implementation of this algorithm todetermine percent identity of a sequence is provided by the GeneticsComputer Group (Madison, Wis.) in the “BestFit” utility application. Thedefault parameters for this method are described in the WisconsinSequence Analysis Package Program Manual, Version 8 (1995) (availablefrom Genetics Computer Group, Madison, Wis.). A preferred method ofestablishing percent identity in the context of the present invention isto use the MPSRCH package of programs copyrighted by the University ofEdinburgh, developed by John F. Collins and Shane S. Sturrok, anddistributed by IntelliGenetics, Inc. (Mountain View, Calif.). From thissuite of packages the Smith-Waterman algorithm can be employed wheredefault parameters are used for the scoring table (for example, gap openpenalty of 12, gap extension penalty of one, and a gap of six). From thedata generated the “Match” value reflects “sequence identity.” Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art, for example, anotheralignment program is BLAST, used with default parameters. For example,BLASTN and BLASTP can be used using the following default parameters:genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+ GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://wvw.ncbi.nlm.qov/cqi-bin/BLAST.

In some embodiments, the heterologous nucleic acid encodes antigenicdomains or antigenic protein fragments rather than the entire antigenicprotein. These fragments can be of any length sufficient to be antigenicor immunogenic. Fragments can be at least 8 amino acids long, preferably10-20 amino acids, but can be longer, such as, e.g., at least 50, 100,200, 500, 600, 800, 1000, 1200, 1600, 2000 amino acids long, or anylength in between.

In some embodiments, at least one nucleic acid fragment encoding anantigenic protein fragment or immunogenic polypeptide thereof isinserted into the viral vector of the invention. In another embodiment,about 2-6 different nucleic acids encoding different antigenic proteinsare inserted into one or more of the viral vectors. In some embodiments,multiple immunogenic fragments or subunits of various proteins can beused. For example, several different epitopes from different sites of asingle protein or from different proteins of the same strain, or from aprotein orthologue from different strains can be expressed from thevectors.

Definitions

It must be noted that, as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “an antigenic determinant”includes one or more antigenic determinants and reference to “themethod” includes reference to equivalent steps and methods known tothose of ordinary skill in the art that could be modified or substitutedfor the methods described herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”. Any of the aforementioned terms (comprising, containing,including, having), whenever used herein in the context of an aspect orembodiment of the present invention may be substituted with the term“consisting of”, though less preferred.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. To the extent the material incorporated byreference contradicts or is inconsistent with this specification, thespecification will supersede any such material. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

The term “substantially similar” in the context of the filovirusantigenic proteins of the invention indicates that a polypeptidecomprises a sequence with at least 90%, preferably at least 95% sequenceidentity to the reference sequence over a comparison window of 10-20amino acids. Percentage of sequence identity is determined by comparingtwo optimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “subtype” herein can be replaced with “species”. It includesstrains, isolates, clades or variants of any filovirus such as Marburgor Ebola virus. The terms “strain” “clade” or “isolate” are technicalterms, well known to the practitioner, referring to the taxonomy ofmicroorganisms. The taxonomic system classifies all so far characterisedmicroorganisms into the hierarchic order of Families, Genera, Species,Strains (Fields Virology, ed. by Fields B. N., Lippincott-RavenPublishers, 4th edition 2001). While the criteria for the members of aFamily is their phylogenetic relationship, a Genera comprises allmembers which share common characteristics, and a Species is defined asa polythetic class that constitutes a replicating lineage and occupies aparticular ecological niche. The term “strain” or “clade” describes amicroorganism, i.e., virus, which shares common characteristics, likebasic morphology or genome structure and organization, but varies inbiological properties, like host range, tissue tropism, geographicdistribution, attenuation or pathogenicity. For example there are fiveEbola virus subtypes known, i.e., Zaire Ebola virus, Sudan Ebola virus,Reston Ebola virus, Bundibugyo Ebola virus and Ivory Coast Ebola virus.Zaire Ebola virus strains are for example Zaire-Mayinga, Zaire-Kikwit,Zaire-Gabon (1994), Zaire-Gabon (February 1996), Zaire-Gabon (October1996). There is only one Marburg virus subtype or species i.e., LakeVictoria marburgvirus know so far with the strains includingMarburg-Musoke and Marburg-Angola. For further strains or isolates seealso FIG. 1.

The term “TCID₅₀” is the abbreviation of “tissue culture infectiousdose”, that amount of a pathogenic agent that will produce pathologicalchange in 50% of cell cultures inoculated, expressed as TCID₅₀/ml. Amethod for determining TCID₅₀ is well known to the person skilled in theart. It is for example described in e.g., Example 2 of WO 03/053463.

The term “subject” as used herein is a living multi-cellular vertebrateorganisms, including, for example, humans, non-human mammals and(non-human) primates. The term “subject” may be used interchangeablywith the term “animal” herein.

The term “filovirus-caused disease” referred to in any of theembodiments can be any disease caused by an infection of any filovirusstrain, isolate or variant thereof as mentioned herein or anycombination of any filovirus strain, isolate or variant (as mentionedanywhere supra or infra and/or in any of the embodiments supra or infra)thereof.

As used herein, the term “enhanced” when used with respect to an immuneresponse against a filovirus, such as an antibody response (e.g.,neutralizing antigen specific antibody response or ZEBOV-GP-specificantibody response), a cytokine response or a CD8 T cell response (e.g.,immunodominant CD8 T cell response), refers to an increase in the immuneresponse in an animal administered with a homologous prime-boostcombination vaccine of MVA relative to the corresponding immune responseobserved from the animal administered with a homologous prime-boostcombination vaccine of MVA vectors, wherein the MVA vectors do notexpress any filovirus virion protein 40 or refers to an increase in theimmune response in an animal administered with a heterologousprime-boost combination vaccine of MVA and FPV vectors according to theinvention, relative to the corresponding immune response observed fromthe animal administered with a heterologous prime-boost combinationvaccine of MVA and FPV vectors according to the invention, wherein theMVA vector does not express any filovirus virion protein 40. Preferably,“enhanced” when used with respect to an immune response, such as anantibody response e.g., neutralizing antibody response, a cytokineresponse or a CD8 T cell response, refers to an increase in the immuneresponse in an animal administered with a heterologous prime-boostcombination vaccine of MVA as a prime and FPV vectors as boost accordingto the invention, relative to the corresponding immune response observedfrom the animal administered with a reverse prime-boost combination,wherein the FPV vector is provided as a prime and the MVA vector isprovided to boost the immune response, using the same prime-boostinterval.

In the context of this invention, an “immunodominant CD8 T cellresponse” means the major CD8 T cell response of a host against arecombinant antigen encoded by a MVA and/or FPV vector. Thus, animmunodominant CD8 T cell response against a recombinant antigen encodedby a homologous prime-boost of recombinant MVA or heterologousprime-boost of recombinant MVA and FPV can be generated that is greaterthan the CD8 T cell response against any recombinant antigen of therecombinant MVA or FPV, wherein the MVA vector does not express anyfilovirus virion protein 40.

The level of the CD8 T cell response can be determined by methods wellknown in the art such as but not limited to an ELISPOT assay (e.g.,interferon gamma (IFN-γ) ELISPOT. Protocols are for examples describedin Current Protocols in Immunology (John Wiley & Son, Inc. (1994) (see,e.g., Chapter 6, Section 19: ELISPOPT Assay to Detect Cytokine-secretingMurine and Human Cells, Supplement 10) or by Schneider, et al., Nat.Med. 4:397-402 (1998)) and, for example, by the techniques set forth inthe examples for a specific virus of the invention. Other suitableassays comprise an ICS assay, which analyzes levels of intracellularcytokine for CD8 T cell activity. For example, the CD8 T cell responsecan comprise an antigen specific CD8 T cell response that is more than50%, such as 51%, 60%, 70%, 80%, 90% or 100% of the total antigenspecific T-cell responses in the animal subject. Preferably, the CD8 Tcell response also represents 0.1% or more, such as 0.1%, 0.2%, 0.3%,0.4%, 0.5%, or more of the total cytokine responses in the animalsubject. In some embodiments, after the second or third boost, therecombinant viral vectors according to the invention induce a CD8 T cellresponse in the host against the encoded antigen that is at least 0.5%,1%, 5%, 10%, 15%, 20%, 25%, or 30% of the total CD8 T cell compartment.

The level of antibody responses can be determined by methods known inthe art. Any suitable plaque reduction neutralization titer (PRNT) assaycan be used to determine whether a polypeptide (or polynucleotideexpressing such a polypeptide) induces one or more neutralizingantibodies against one or more filovirus antigens of one or morefilovirus subtype. An exemplary plaque reduction neutralization titerassay for filoviruses is described in the examples. Other PRNT methodsand formats are well known to those of ordinary skill in the art.

Filovirus Proteins

As used interchangeably herein, the terms “glycoprotein gene” or “GPgene” refer to the gene, or to a homologue or variant of the gene,encoding the glycoprotein, in particular the transmembrane envelopeglycoprotein, in any filovirus strain or isolate, even though the exactsequence and/or genomic location of the glycoprotein gene may differbetween strains or isolates. For example, in the Maleo strain of SEBOV(SEBOV-Maleo), the glycoprotein gene (GP-SEBOV-Maleo gene) comprisesnucleotides 120-1004 and 1004-2149 (endpoints included) as numbered inGenBank Accession Number U23069.1. The EBOV transcripts undergo editingduring transcription such that some nucleotides are read twice. TheGP-SEBOV-Maleo gene further comprises a protein coding open readingframe (ORF) spanning nucleotides 120-1004 and 1004-2149 (endpointsincluded) as numbered in GenBank Accession Number U23069.1. Thenucleotide sequence of the GP-SEBOV-Maleo gene is set forth in SEQ IDNO:1 (GenBank Accession No. U23069.1).

As used herein, a “homologue” or “variant” preferably has at least about50%, at least about 60% or 65%, at least about 70% or 75%, at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, moretypically, at least about 90%, 91%, 92%, 93%, or 94% and even moretypically at least about 95%, 96%, 97%, 98% or 99%, most typically, atleast about 99% nucleotide sequence identity with the referenced gene,protein, polypeptide, antigenic protein fragment, antigen and epitope.The term “homologue” or “variant” also encompasses deleted, truncated orotherwise mutated versions of the genes and proteins, respectively. Byway of example, encompassed are, e.g., soluble forms of the GP-EBOV orGP-MARV proteins lacking the signal peptide as well as the transmembraneand/or cytoplasmic domains of the full-length GP-EBOV or GP-MARVproteins.

As used interchangeably herein, the terms “glycoprotein” or “GP” referto the glycoprotein, in particular the transmembrane envelopeglycoprotein, or to a homologue or variant of the glycoprotein.

The amino acid sequence of GP-EBOV-Maleo is set forth in SEQ ID NO:2(amino acid sequence of GenBank Accession No. U23069.1). TheGP-SEBOV-Maleo protein comprises a signal peptide, an extracellulardomain, a transmembrane domain, and a cytoplasmic domain (see, e.g.,UniProtKB/Swiss-Prot Accession No. Q66798). The signal peptide ofGP-SEBOV-Maleo protein consists of amino acids 1-32 of SEQ ID NO:2; theextracellular domain of GP-SEBOV-Maleo protein consists of amino acids33-650 of SEQ ID NO:2 or amino acids 1-650 of SEQ ID NO:2; thetransmembrane domain of GP-SEBOV-Maleo protein consists of amino acids651-671 of SEQ ID NO:2; and the cytoplasmic domain of GP-SEBOV-Maleoprotein consists of amino acids 672-676 of SEQ ID NO:2.

The nucleic acid encoding the amino acid sequence of GP-ZEBOV-Mayinga isset forth in SEQ ID NO:19. The GP-ZEBOV-Mayinga comprises a protein asset forth in SEQ ID NO:20 (GenBank Accession Number ABX75367.1).

Likewise, also the terms “nucleoprotein gene” or “NP gene”, as usedinterchangeably herein, refer to the gene, or to a homologue or variantof the gene, encoding the nucleoprotein in any filovirus strain orisolate, even though the exact sequence and/or genomic location of thenucleoprotein gene may also differ between strains or isolates. Forexample, in the Boniface strain of SEBOV (SEBOV-Boniface), thenucleoprotein gene (NP-SEBOV-Boniface gene) comprises nucleotides383-2599 (endpoints included) as numbered in GenBank Accession NumberAF173836.1. The NP-SEBOV-Boniface gene further comprises a proteincoding open reading frame (ORF) spanning nucleotides 383-2599 (endpointsincluded) as numbered in GenBank Accession Number AF173836.1. Thenucleotide sequence of the NP-SEBOV-Boniface gene is set forth in SEQ IDNO:3 (GenBank Accession No. AF173836.1).

The amino acid sequence of NP-EBOV-Boniface is set forth in SEQ ID NO:4(amino acid sequence of GenBank Accession No. AF173836.1). TheNP-SEBOV-Boniface protein comprises a coiled coil domain (see, e.g.,UniProtKB/Swiss-Prot Accession No. Q9QP77). The coiled coil domain ofNP-SEBOV-Boniface protein consists of amino acids 334-363 of SEQ IDNO:4.

In certain embodiments, the nucleic acid encoding an antigenicdeterminant, preferably an antigenic protein, more preferably of any ofthe proteins as mentioned supra or infra is a full-length protein.

Recombinant MVA and FPV

Provided herein are recombinant poxviruses (e.g., MVA or MVA-BN or FPV)comprising heterologous or foreign nucleic acid sequences derived fromEBOV and/or MARV incorporated in a variety of insertion sites in thepoxviral (e.g., MVA or MVA-BN or FPV) genome. The heterologous nucleicacids can encode one or more foreign proteins and/or foreign antigensincluding, for example, viral antigens.

Generally, a “recombinant” MVA or FPV as described herein refers toMVAs/FPVs that are produced by standard genetic engineering methods,i.e., MVAs/FPVs of the present invention are thus genetically engineeredor genetically modified MVAs/FPCs. The term “recombinant MVA or FPV”thus includes MVAs/FPVs which have stably integrated recombinant nucleicacid, preferably in the form of a transcriptional unit, in their genome.A transcriptional unit may include a promoter, enhancer, terminatorand/or silencer. Recombinant MVAs/FPVs of the present invention mayexpress heterologous antigenic determinants, polypeptides or proteins(antigens) upon induction of the regulatory elements. The term “MVA/FPV”in the context of any of the embodiments of the invention encompassesboth individual and combined options for MVA, FPV or MVA and FPV.

As used herein, a “heterologous” gene, nucleic acid, antigen, or proteinis understood to be a nucleic acid or amino acid sequence which is notpresent in the wild-type poxviral genome (e.g., MVA or MVA-BN or FPV).The skilled person understands that a “heterologous gene”, when presentin a poxvirus such as MVA or MVA-BN or FPV, is to be incorporated intothe poxviral genome in such a way that, following administration of therecombinant poxvirus to a host cell, it is expressed as thecorresponding heterologous gene product, i.e., as the “heterologousantigen” and/or “heterologous protein.” Expression is normally achievedby operatively linking the heterologous gene to regulatory elements thatallow expression in the poxvirus-infected cell. Preferably, theregulatory elements include a natural or synthetic poxviral promoter.

In one aspect, the recombinant MVA/FPV vector according to the inventioncomprises a heterologous nucleotide sequence encoding an antigenicdeterminant of a filovirus protein selected from an Ebola virus (EBOV)and/or a Marburg virus (MARV). In another embodiment, the recombinantMVA/FPV vector according to the invention comprises a heterologousnucleotide sequence encoding an antigenic determinant of one or moreantigenic determinant(s) of the filovirus protein (e.g., EBOV protein)which is selected from one or more EBOV subtypes selected from the groupconsisting of Zaire Ebola virus (ZEBOV), Sudan Ebola virus (SEBOV), Coted'Ivoire Ebola virus (EBOV-Cdl, also called Tai Forest virus or TAFV),Reston Ebola virus (REBOV) and Bundibugyo Ebola virus (BEBOV).

According to another embodiment, the recombinant MVA/FPV vectoraccording to the invention comprises one or more antigenicdeterminant(s) of a filovirus protein, preferably EBOV protein, MARVprotein or full-length protein thereof, selected from the group ofZaire-Mayinga, Zaire-Kikwit, Zaire-Gabon, Cote d'Ivoire Ebola virus,Sudan-Boniface, Sudan-Maleo, Sudan-Gulu, Marburg-Ravn, Marburg-Ozolin,Marburg-Ratayczak, Marburg-Musoke, Marburg-Angola.

Preferably, the antigenic determinant of the filovirus protein (e.g.,selected from the group of Zaire-Mayinga, Zaire-Kikwit, Zaire-Gabon,Cote d'Ivoire Ebola virus, Sudan-Boniface, Sudan-Maleo, Sudan-Gulu,Marburg-Ravn, Marburg-Ozolin, Marburg-Ratayczak, Marburg-Musoke,Marburg-Angola) is selected from the group consisting of an envelopeglycoprotein (GP), nucleoprotein (NP), virion protein 35 (VP35), virionprotein 40 (VP40), virion protein 30 (VP30), virion protein 24 (VP24),and RNA-directed RNA polymerase protein (L).

In another embodiment, the antigenic determinant of the filovirusprotein is an envelope glycoprotein (GP), preferably at least anenvelope glycoprotein (GP) and a virion protein 40 (VP40).

In another embodiment, the antigenic determinant of the filovirusprotein is an envelope glycoprotein (GP) selected from the group ofZEVOV and SEBOV, preferably at least an envelope glycoprotein (GP) and avirion protein 40 (VP40), wherein the GP and VP40 are derived from thesame strain, preferably wherein the same strain is selected from thegroup of ZEBOV and SEBOV.

In another embodiment, the antigenic determinant of the filovirusprotein is at least an envelope glycoprotein (GP) and a virion protein40 (VP40), wherein the GP and VP40 are derived from a different isolateor the same isolate, preferably wherein the different or the sameisolate is selected from the group of Zaire-Mayinga, Zaire-Kikwit,Zaire-Gabon, Cote d'Ivoire Ebola virus, Sudan-Boniface, Sudan-Maleo,Sudan-Gulu, Marburg-Ravn, Marburg-Ozolin, Marburg-Ratayczak,Marburg-Musoke and Marburg-Angola, preferably wherein the isolate isselected from the group of Zaire-Mayinga, Sudan-Gulu, Marburg-Musoke andMarburg-Angola, most preferably wherein the isolate is selected from thegroup of Zaire-Mayinga, Sudan-Gulu and Marburg-Musoke.

In another preferred embodiment, the recombinant MVA/FPV vectoraccording to the invention comprises a nucleotide sequence encoding anantigenic determinant of two, three, four or more Ebola and/or Marburgsubtypes.

Another preferred embodiment covers the recombinant MVA/FPV vectoraccording to any of the embodiments of the invention which comprises anantigenic determinant of two, three, four or more filovirus proteinsselected from the group consisting of envelope glycoprotein (GP),nucleoprotein (NP), virion protein 35 (VP35), virion protein 40 (VP40),virion protein 30 (VP30), virion protein 24 (VP24), and RNA-directed RNApolymerase protein (L).

In a preferred embodiment, the recombinant MVA/FPV vector according toany of the embodiments of the invention comprises an antigenicdeterminant of one, two, three, four or more filovirus protein selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:20, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:37.

In a preferred embodiment, the recombinant MVA/FPV vector according toany of the embodiments of the invention comprises an antigenicdeterminant of a filovirus protein selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:29, SEQID NO:31, SEQ ID NO:34 and SEQ ID NO:37.

In another embodiment, the recombinant MVA/FPV vector according to anyof the embodiments of the invention comprises an antigenic determinantof a filovirus protein consisting of SEQ ID NO: 20.

In another embodiment, the recombinant MVA/FPV vector according to anyof the embodiments of the invention comprises an antigenic determinantof a filovirus protein selected from the group consisting of SEQ ID NO:20 and SEQ ID NO: 34.

In another embodiment, the recombinant MVA/FPV vector according to anyof the embodiments of the invention comprises an antigenic determinantof a filovirus protein selected from the group consisting of SEQ IDNO:6, SEQ ID NO:20, SEQ ID NO:31 and SEQ ID NO:34.

In another embodiment, the recombinant MVA/FPV vector according to anyof the embodiments of the invention comprises an antigenic determinantof a filovirus protein selected from the group consisting of SEQ IDNO:6, SEQ ID NO: 20, SEQ ID NO:29, SEQ ID NO:31 and SEQ ID NO:34.

In another preferred embodiment, the recombinant MVA/FPV vectoraccording to any of the embodiments of the invention comprises anantigenic determinant of a filovirus protein selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:29, SEQ ID NO:31, SEQID NO:34 and SEQ ID NO:37.

In another preferred embodiment, the MVA vector according to any of theembodiments of the invention comprises a heterologous nucleotidesequence encoding an antigenic determinant of a filovirus proteinconsisting of SEQ ID NO:29 and/or SEQ ID NO:6, SEQ ID NO:20, SEQ IDNO:31.

In another preferred embodiment, the MVA vector according to any of theembodiments of the invention comprises a nucleotide sequence comprisingSEQ ID NO:28 and/or SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:30.

In another preferred embodiment, the MVA vector according to any of theembodiments of the invention comprises a nucleotide sequence encoding anantigenic protein of a filovirus virion protein 40 (VP40) comprising SEQID NO:33 or a nucleotide sequence encoding the protein sequencecomprising SEQ ID NO:34.

In another preferred embodiment, the recombinant MVA vector according toany of the embodiments of the invention comprising a nucleotide sequenceselected from the group of a) SEQ ID NO:5, SEQ ID NO:19 and SEQ IDNO:30, b) SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:28 and SEQ ID NO:30 andc) SEQ ID NO:19 and SEQ ID NO:33.

In another aspect, the present invention comprises a recombinant MVAvector or FPV vector comprising a heterologous nucleotide sequenceencoding an antigenic determinant of a filovirus glycoprotein, inparticular a filovirus envelope glycoprotein.

The filovirus glycoprotein may encode a GP-MARV or a GP-EBOV.

For the embodiments as described herein the glycoprotein of MARV may bederived from MARV-Musoke, preferably the full-length MARV-Musoke, which,in turn, may be derived from the Lake Victoria strain or isolate ofMARV-Musoke. The GP-MARV may also be derived from MARV-Ravn MARV-Ozolin,MARV-Ratayczak or from MARV-Angola. A nucleotide sequence encoding afull-length GP-MARV-Musoke is shown in SEQ ID NO:5 encoding amino acids1 to 681 or 19 to 681 of SEQ ID NO:6. In a preferred embodiment, theGP-MARV-Musoke comprises the nucleotide sequence of SEQ ID NO:5preferably encoding the protein of SEQ ID NO:6. In certain embodiments,the GP-MARV-Musoke is truncated wherein the truncated GP-MARV-Musoke maycomprise only the extracellular domain of the envelope glycoprotein,comprising amino acids 1 to 648 or amino acids 19 to 648 of SEQ ID NO:6(GenBank Accession No. ABA87127.1). In other embodiments as describedherein the glycoprotein of MARV may be derived from MARV-Angola,preferably the full-length GP-MARV-Angola. In a preferred embodiment,the GP-MARV-Angola comprises the nucleotide sequence of SEQ ID NO:36encoding amino acids of SEQ ID NO:37.

The glycoprotein of EBOV may be GP-SEBOV or may be derived fromGP-ZEBOV, in particular from the Mayinga strain of GP-ZEBOV(GP-ZEBOV-Mayinga). The full-length GP-ZEBOV-Mayinga comprises thenucleotide sequence of SEQ ID NO:19 encoding the amino acid sequence ofSEQ ID NO:20. In a preferred embodiment, the GP-ZEBOV-Mayinga comprisesthe nucleotide sequence of SEQ ID NO:19 preferably encoding the proteinof SEQ ID NO:20. The GP-EBOV may also be GP-BEBOV, GP-EBOV-Cdl orGP-EBOV-Reston. The GP-ZEBOV may be truncated and may comprise thenucleotide sequence of SEQ ID NO:19 modified to encode amino acids 1-636of SEQ ID NO:20 or modified to delete the mucin domain spanning aminoacids 314 to 464 of SEQ ID NO:20.

The GP-SEBOV may be derived from the Gulu strain of GP-SEBOV(GP-SEBOV-Gulu). In certain embodiments, the GP-SEBOV comprises thenucleotide sequence of SEQ ID NO:30, preferably encoding the amino acidsequence of SEQ ID NO:31.

The recombinant MVA/FPV according to the present invention can alsofurther comprise tetanus toxoid fragment C sequence. In a preferredembodiment, GP-MARV-Musoke, in particular the full-length MARV-MusokeGP, further comprises tetanus toxoid fragment C. A tetanus toxoidfragment C may comprise the nucleotide sequence of SEQ ID NO:7 encodingthe amino acid sequence of SEQ ID NO:8. In certain embodiments, thetruncated GP-MARV-Musoke further comprises tetanus toxoid fragment C(TTC) which may comprise the nucleotides 2281-3642 of the nucleotidesequence of SEQ ID NO:7 encoding amino acids 760-1213 of the amino acidsequence of SEQ ID NO:8.

The recombinant MVA/FPV vector according to the present invention canadditionally comprise an immunostimulatory or co-stimulatory molecule.In a preferred embodiment, the heterologous nucleotide sequence encodingan antigenic determinant of a GP-MARV-Musoke further comprises one ormore immunostimulatory molecules. In certain embodiments, the one ormore immunostimulatory molecules is human CD40 ligand (hCD40L) which maycomprise SEQ ID NO:9 encoding the amino acid sequence of SEQ ID NO:10.In certain embodiments, the one or more immunostimulatory molecule(s) isa fusion protein comprising the sushi domain of human interleukin-15receptor (hIL15R-Sushi) which may comprise SEQ ID NO:11 encoding theamino acid sequence of SEQ ID NO:12.

The one or more immunostimulatory molecules may also be lymphocytefunction-associated antigen 3 (LFA-3, or CD58), intercellular adhesionmolecule 1 (ICAM-1, or CD54) and B7.1 (CD80), collectively known as thetriad of costimulatory molecules (i.e., ‘TRICOM’). “TRICOM” as usedherein is an abbreviation for Triad of COstimlatory Molecules consistingof B7-1 (also known as CD80), intracellular adhesion molecule-1 (ICAM-1,also known as CD54) and lymphocyte function-associated antigen-3 (LFA-3,also known as CD58), included in the recombinant viral vectors (e.g.,poxviral vectors) expressing a specific antigen in order to increase theantigen-specific immune response. The individual components of TRICOMcan be under the control of the same or different promoters, and can beprovided on the same vector with the specific antigen or on a separatevector. Exemplary vectors are disclosed, for example, in Hodge et al.,“A Triad of Costimulatory Molecules Synergize to Amplify T-CellActivation,” Cancer Res. 59:5800-5807 (1999) and U.S. Pat. No. 7,211,432B2, both of which are incorporated herein by reference. The LFA-3 maycomprise the nucleotide sequence of SEQ ID NO:13 encoding the amino acidsequence of SEQ ID NO:14, the ICAM-1 may comprise the nucleotidesequence of SEQ ID NO:15 encoding the amino acid sequence of SEQ IDNO:16, and the B7.1 may comprise the nucleotide sequence of SEQ ID NO:17encoding the amino acid sequence of SEQ ID NO:18.

The recombinant MVA/FPV according to the present invention may alsoadditionally comprise a membrane anchor sequence such as the vacciniavirus gene B5m comprising the nucleotide sequence of SEQ ID NO:21encoding the amino acid sequence of SEQ ID NO:22. In particular, theantigenic determinant as described herein may preferably be operablylinked to a membrane anchor such as the B5m. Thus, when used herein thatthe recombinant MVA/FPV comprises a membrane anchor sequence, it ismeant that an antigenic determinant comprised by the recombinant MVA/FPVis preferably operably linked to the membrane anchor. A membrane anchorrefers to any polypeptide capable of anchoring heterologous polypeptidesto the outer face of the cell membrane. Preferably, the membrane anchorcomprises the cytoplasmic and transmembrane domains of Vaccinia virusB5R protein, termed herein as the “B5R anchor” or “B5m” As defined, aB5R anchor refers to the 42-amino-acid C-terminal segment of the B5Rprotein from any type of Vaccinia virus, for example, the WR strain(Katz et al. J Virol. 71(4):3178-87 (1997)) or more preferably a MVA. Inaddition, B5R anchor variants having at least 80%, such as at least 85%,for example at least 90%, or at least 95%, such as at least 98% sequenceidentity with respect to the reference B5R anchor sequence are alsoincluded in the present invention. A preferred anchor sequence is shownin SEQ ID NO: 21, its translation product is also shown in SEQ ID NO:22.

In a preferred embodiment, the full-length and/or truncated GP-ZEBOVfurther comprises vaccinia virus gene B5m.

In another aspect, the present invention comprises a recombinant MVA/FPVvector comprising a heterologous nucleotide sequence encoding anantigenic determinant of a filovirus glycoprotein as described above,and further comprises heterologous nucleotide sequences encodingadditional filovirus proteins required to form virus-like particles(VLP). In one embodiment, the additional heterologous nucleotidesequence encoding filovirus protein required to form VLPs can be VP40.In certain embodiments, the additional filovirus proteins required toform virus-like particles or enhancing formation of VLPs are NP-EBOV andVP40-EBOV wherein these proteins may be derived from the strains asindicated above. Preferably, the filovirus nucleoprotein (e.g., NP-EBOV)and a filovirus virion protein 40 (e.g., VP40-EBOV) are derived from thesame filovirus strain. By vaccinating non-human primates with arecombinant MVA expressing GP and VP40 (either in addition or withoutexpressing NP) and which is capable of generating GP-containingEBOV-VLPs from infected cells the inventors could achieve protectionagainst filovirus challenge in non-human primates. The production ofvirus-like particles in the animals being vaccinated creates anadditional vaccine modality closely mimicking the viral particlespresent in a bona fide filoviral infection. Such recombinant MVA filoVLP vaccination stimulated both the humoral and cellular immune responseand thus protected against filovirus challenge. A further advantage ofvaccination with an attenuated MVA virus providing filoviral VLPs is tocircumvent the need for purification of virus-like particles forinoculation and the additional MVA mediated immune stimulation. The useof a filovirus nucleoprotein (e.g., NP-EBOV) and a filovirus virionprotein 40 (e.g., VP40-EBOV) derived from the same strain is ofadvantage for enhancing the formation of VLPs, preferably for generatinghomogenous GP spike decorated VLPs with a homogenous diameter forclosely mimicking the viral particles and improving protection against afilovirus infection.

The present invention also relates to a recombinant MVA/FPV vectorcomprising a heterologous nucleotide sequence encoding an antigenicdeterminant of a filovirus glycoprotein and a heterologous nucleotidesequence encoding an antigenic determinant of a further filovirusprotein. The nucleotide sequence encoding an antigenic determinant of afurther filovirus protein may encode one or more filovirus proteinsselected from the group consisting of nucleoprotein (NP), virion protein35 (VP35), virion protein 40 (VP40), virion protein 30 (VP30), virionprotein 24 (VP24), and RNA-directed RNA polymerase protein (L). Saidgenes and proteins, respectively, can be derived from the one or morefilovirus strains described above. The NP-EBOV-Cdl of certainembodiments comprises the nucleotide sequence of SEQ ID NO:28 encodingthe amino acid sequence of SEQ ID NO:29.

In certain embodiments, VP40 is selected from MARV or EBOV, preferablyVP40 is selected from one or more EBOV subtypes selected from the groupconsisting of Zaire Ebola virus (ZEBOV), Sudan Ebola virus (SEBOV), Coted'Ivoire Ebola virus (EBOV-Cdl, also called Tai Forest virus or TAFV),Reston Ebola virus (EBOV-Reston) and Bundibugyo Ebola virus (BEBOV). Ina preferred embodiment, VP40 is selected from one or more of ZEBOV,SEBOV and MARV. In certain embodiments, the filovirus glycoprotein andthe filovirus VP40 are selected from the same filovirus strain. In afurther preferred embodiment, VP40 and/or the filovirus glycoprotein areselected from one or more of Zaire-Mayinga, Zaire-Kikwit, Zaire-Gabon,Cote d'Ivoire Ebola virus, Sudan-Boniface, Sudan-Maleo, Sudan-Gulu,Marburg-Ravn, Marburg-Ozolin, Marburg-Ratayczak, Marburg-Musoke andMarburg-Angola, more preferably selected from one or more ofZaire-Mayinga (VP40-ZEBOV-Mayinga), Sudan-Gulu (VP40-SEBOV-Gulu),Marburg-Musoke (VP40-MARV-Musoke) and Marburg-Angola (VP40-MARV-Angola).In a further embodiment, the MVA vector of any of the embodimentsfurther comprises a filovirus nucleoprotein (NP), preferably wherein thefilovirus nucleoprotein and the filovirus VP40 are derived from the samefilovirus strain. In a further embodiment, VP40 comprises the nucleicsequence encoding VP40-ZEBOV-Mayinga or VP40-MARV-Musoke. In otherembodiments, filovirus VP40 comprises the nucleotide sequence of SEQ IDNO:33. In a further embodiment, VP40 comprises a nucleic acid encodingthe protein sequence of SEQ ID NO:34. In a further preferred embodiment,VP40 comprises the nucleotide sequence of SEQ ID NO:33 encoding theamino acid sequence of SEQ ID NO:34.

In a further preferred embodiment, the recombinant MVA/FPV vectorcomprises two heterologous nucleotide sequences encoding an antigenicdeterminant of a filovirus envelope glycoprotein and at least oneheterologous nucleotide sequence encoding an antigenic determinant of afurther filovirus protein. In certain embodiments, the firstheterologous nucleotide sequence encoding an antigenic determinant of afilovirus envelope glycoprotein encodes a GP-MARV, and the secondheterologous nucleotide sequence encoding an antigenic determinant of afilovirus envelope glycoprotein encodes a GP-EBOV. The recombinantMVA/FPV vector comprises, according to a further preferred embodiment ofthe present invention, three heterologous nucleotide sequences encodingan antigenic determinant of a filovirus envelope glycoprotein and atleast one heterologous nucleotide sequence encoding an antigenicdeterminant of a further filovirus protein. Preferably, the firstheterologous nucleotide sequence encoding an antigenic determinant of afilovirus envelope glycoprotein encodes a GP-MARV, the secondheterologous nucleotide sequence encoding an antigenic determinant of afilovirus envelope glycoprotein encodes a GP-EBOV, and the thirdheterologous nucleotide sequence encoding an antigenic determinant of afilovirus envelope glycoprotein encodes a GP-EBOV derived from an EBOVstrain or isolate different than the GP-EBOV encoded by the secondheterologous nucleotide sequence. Accordingly, one heterologousnucleotide sequence encoding an antigenic determinant of a filovirusenvelope glycoprotein may encode GP-SEBOV-Gulu and the other oneGP-ZEBOV-Mayinga.

In another embodiment, the recombinant MVA/FPV vector comprises twoheterologous nucleotide sequences encoding an antigenic determinant of aGP-EBOV from an EBOV strain or isolate and two heterologous nucleotidesequence encoding an antigenic determinant of a GP-MARV from an MARVstrain or isolate, preferably the MARV strain is MARV-Angola andMARV-Musoke and the EBOV strain is ZEBOV and/or SEBOV, preferablyZEBOV-Mayinga and SEBOV-Gulu. Of course, the further nucleotide sequenceencoding an antigenic determinant of a further filovirus protein mayencode also filovirus proteins selected from the group consisting ofnucleoprotein (NP), virion protein 35 (VP35), virion protein 40 (VP40),virion protein 30 (VP30), virion protein 24 (VP24), and RNA-directed RNApolymerase protein (L), as already mentioned above which may also bederived from the different strains as already indicated above.

The recombinant MVA/FPV vector according to a further preferredembodiment of the present invention comprises two heterologousnucleotide sequences encoding an antigenic determinant of a filovirusenvelope glycoprotein of GP-MARV and GP-EBOV and a third heterologousnucleotide sequence encoding an antigenic determinant of VP40. Such VP40can be any of the VP40 as described supra or infra. Accordingly oneheterologous nucleotide sequence encoding an antigenic determinant of afilovirus envelope glycoprotein may encode GP-SEBOV-Gulu, the other oneGP-ZEBOV-Mayinga and the third heterologous nucleotide sequence mayencode an antigenic determinant of filovirus protein VP40-ZEBOV,VP40-SEBOV or VP40-MARV, preferably VP40-ZEBOV-Mayinga orVP40-MARV-Musoke.

In a further preferred embodiment, the recombinant MVA/FPV vectorcomprises two heterologous nucleotide sequences encoding an antigenicdeterminant of a filovirus envelope glycoprotein GP-EBOV, preferablyGP-ZEBOV and/or GP-SEBOV, more preferably GP-ZEBOV-Mayinga andGP-SEBOV-Gulu, one filovirus envelope glycoprotein of GP-MARV,preferably GP-MARV-Musoke or GP-MARV-Angola and at least one filovirusnucleoprotein, preferably selected from the group of NP-EBOV-Cdl,NP-ZEBOV and NP-MARV, preferably NP-MARV-Musoke or NP-MARV-Angola.

Integration Sites into MVA/FPV

Heterologous nucleotide sequences encoding antigenic determinants of afilovirus glycoprotein, optionally further comprising at least oneheterologous nucleotide sequence encoding a further filovirus proteinmay be inserted into one or more intergenic regions (IGR) of the MVA. Incertain embodiments, the IGR is selected from IGR07/08, IGR 44/45, IGR64/65, IGR 88/89, IGR 136/137, and IGR 148/149. In certain embodiments,less than 5, 4, 3, or 2 IGRs of the recombinant MVA compriseheterologous nucleotide sequences encoding antigenic determinants of afilovirus envelope glycoprotein and/or a further filovirus protein. Theheterologous nucleotide sequences may, additionally or alternatively, beinserted into one or more of the naturally occurring deletion sites, inparticular into the main deletion sites I, II, III, IV, V, or VI of theMVA genome. In certain embodiments, less than 5, 4, 3, or 2 of thenaturally occurring deletion sites of the recombinant MVA compriseheterologous nucleotide sequences encoding antigenic determinants of afilovirus envelope glycoprotein and/or a further filovirus protein.

The number of insertion sites of MVA comprising heterologous nucleotidesequences encoding antigenic determinants of a filovirus protein can be1, 2, 3, 4, 5, 6, 7, or more. In certain embodiments, the heterologousnucleotide sequences are inserted into 4, 3, 2, or fewer insertionsites. Preferably, two insertion sites are used. In certain embodiments,three insertion sites are used. Preferably, the recombinant MVAcomprises at least 2, 3, 4, 5, 6, or 7 genes inserted into 2 or 3insertion sites.

Heterologous nucleotide sequences encoding antigenic determinants of afilovirus glycoprotein, optionally further comprising at least oneheterologous nucleotide sequence encoding a further filovirus proteinmay be inserted into one or more intergenic regions (IGR) of the FPV. Ina preferred embodiment, the IGR is situated between ORFs 7 and 9 of the1.3-kbp HindIII fragment of the genome (see Drillien et al, Virology160:203-209 (1987) (U.S. Pat. No. 5,180,675) and Spehner et al, J.Virol. 64:527-533 (1990)). In certain embodiments, heterologousnucleotide sequences may be inserted in fowlpox insertion sites asdescribed in EP 0 538 496 A1 and WO 05/048957 incorporated by referenceherewith. Also preferred fowlpox insertion sites of the presentinvention are the LUS insertion site, the FP14 insertion site, and the43K insertion site. These sites are also referred to sometimes asFPN006/FPN007 (LUS insertion site), FPN254/FPN255 (LUS insertion site),FPV060/FPV061 (FP14 insertion site), and FPV107/FPV108 (43K insertionsite).

In one preferred embodiment, the insertion site in fowlpox is the LUSinsertion site. There are two long unique sequences (LUS) at each end ofthe fowlpox viral genome (Genbank Accession NO: AF 198100.1), and thustwo LUS insertion sites in each genome. The LUS insertion site at theleft end of the genome lies 3′ of FPV006 and 5′ of FPV007 125L,preferably between position 7470 and 7475 in the fowlpox genomicsequence as annotated in GenBank Accession No. AF198100.1. The LUSinsertion site at the right end of the genome lies 3′ of FPV254 and 5′of FPV255, preferably between position 281065 and 281070 in the fowlpoxgenomic sequence e.g., of GenBank Accession No. AF198100.1. In oneembodiment, the heterologous nucleotide sequence can be inserted at anyposition within the nucleotide position 281065 and 281070.

In another preferred embodiment, the insertion site in fowlpox is theFP14 insertion site. This site lies 3′ of FPV060 and 5′ of FPV061 in thefowlpox genomic sequence, preferably between position 67080 and 67097 ofthe fowlpox genome e.g., of GenBank Accession No. AF198100.1. In oneembodiment, the nucleotides between position 67080 and 67097 of the DNAsequence are deleted in the recombinant virus and replaced with definedinserts representing a sequence of interest. In one embodiment, the FP14insertion site is between the orthologue of the FPV060 gene and theorthologue of FPV061 e.g., of AF198100.1. The term “FPV060, FPV061,FPV254” etc. refers to the position of the corresponding coding sequence(i.e., CDS) of the respective gene numbered from 5′ to 3′ as annotatedin GenBank Accession No. AF198100.1. In a preferred embodiment, the FP14insertion site is between position 67091 and 67092 in the fowlpoxgenomic sequence (referred to also as IGR60/61 insertion site asannotated in GenBank Accession No. AF198100.1).

In yet another preferred embodiment, the insertion site in fowlpox isdesignated the 43K insertion site. This site lies 3′ of FPV107 and 5′ ofFPV108, preferably at position 128178 of the fowlpox genomic sequence asannotated in GenBank Accession No. AF198100.1.

In a preferred embodiment, the integration site is FP14 (IGR60/61)and/or the BamHI J region. The BamH1 J region is further described in S.Jenkins et al. (1991), Aids Research and Human Retroviruses7(12):991:998 incorporated by reference herewith.

In a certain embodiment, the IGR is IGR BamHI J FPV.

The number of insertion sites of the FPV comprising heterologousnucleotide sequences encoding antigenic determinants of a filovirusprotein can be one or two. Preferably, two insertion sites are used. Inanother preferred embodiment, the recombinant FPV comprises at least 1,2, 3, 4 or 5 genes inserted into one or two insertion sites.

The recombinant MVA/FPV viruses provided herein can be generated byroutine methods known in the art. Methods to obtain recombinantpoxviruses or to insert exogenous coding sequences into a poxviralgenome are well known to the person skilled in the art. For example,methods for standard molecular biology techniques such as cloning ofDNA, DNA and RNA isolation, Western blot analysis, RT-PCR and PCRamplification techniques are described in Molecular Cloning, Alaboratory Manual (2nd Ed.) (J. Sambrook et al., Cold Spring HarborLaboratory Press (1989)), and techniques for the handling andmanipulation of viruses are described in Virology Methods Manual (B. W.J. Mahy et al. (eds.), Academic Press (1996)). Similarly, techniques andknow-how for the handling, manipulation and genetic engineering of MVAare described in Molecular Virology: A Practical Approach (A. J. Davison& R. M. Elliott (Eds.), The Practical Approach Series, IRL Press atOxford University Press, Oxford, UK (1993) (see, e.g., Chapter 9:Expression of genes by Vaccinia virus vectors)) and Current Protocols inMolecular Biology (John Wiley & Son, Inc. (1998) (see, e.g., Chapter 16,Section IV: Expression of proteins in mammalian cells using vacciniaviral vector)).

For the generation of the various recombinant MVAs/FPVs disclosedherein, different methods may be applicable. The DNA sequence to beinserted into the virus can be placed into an E. coli plasmid constructinto which DNA homologous to a section of DNA of the MVA/FPV has beeninserted. Separately, the DNA sequence to be inserted can be ligated toa promoter. The promoter-gene linkage can be positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of MVA/FPV DNAcontaining a non-essential locus. The resulting plasmid construct can beamplified by propagation within E. coli bacteria and isolated. Theisolated plasmid containing the DNA gene sequence to be inserted can betransfected into a cell culture, e.g., of chicken embryo fibroblasts(CEFs), at the same time the culture is infected with MVA. Recombinationbetween homologous MVA DNA in the plasmid and the viral genome,respectively, can generate a MVA modified by the presence of foreign DNAsequences.

According to a preferred embodiment, a cell of a suitable cell cultureas, e.g., CEF cells, can be infected with a poxvirus. The infected cellcan be, subsequently, transfected with a first plasmid vector comprisinga foreign or heterologous gene or genes, preferably under thetranscriptional control of a poxvirus expression control element. Asexplained above, the plasmid vector also comprises sequences capable ofdirecting the insertion of the exogenous sequence into a selected partof the poxviral genome. Optionally, the plasmid vector also contains acassette comprising a marker and/or selection gene operably linked to apoxviral promoter. Suitable marker or selection genes are, e.g., thegenes encoding the green fluorescent protein, β-galactosidase,neomycin-phosphoribosyltransferase or other markers. The use ofselection or marker cassettes simplifies the identification andisolation of the generated recombinant poxvirus. However, a recombinantpoxvirus can also be identified by PCR technology. Subsequently, afurther cell can be infected with the recombinant poxvirus obtained asdescribed above and transfected with a second vector comprising a secondforeign or heterologous gene or genes. In case, this gene shall beintroduced into a different insertion site of the poxviral genome, thesecond vector also differs in the poxvirus-homologous sequencesdirecting the integration of the second foreign gene or genes into thegenome of the poxvirus. After homologous recombination has occurred, therecombinant virus comprising two or more foreign or heterologous genescan be isolated. For introducing additional foreign genes into therecombinant virus, the steps of infection and transfection can berepeated by using the recombinant virus isolated in previous steps forinfection and by using a further vector comprising a further foreigngene or genes for transfection.

Alternatively, the steps of infection and transfection as describedabove are interchangeable, i.e., a suitable cell can at first betransfected by the plasmid vector comprising the foreign gene and, then,infected with the poxvirus. As a further alternative, it is alsopossible to introduce each foreign gene into different viruses, coinfecta cell with all the obtained recombinant viruses and screen for arecombinant including all foreign genes. A third alternative is ligationof DNA genome and foreign sequences in vitro and reconstitution of therecombined vaccinia virus DNA genome using a helper virus. A fourthalternative is homologous recombination in E. coli or another bacterialspecies between a vaccinia virus genome cloned as a bacterial artificialchromosome (BAC) and a linear foreign sequence flanked with DNAsequences homologous to sequences flanking the desired site ofintegration in the vaccinia virus genome.

Expression of Heterologous Filovirus Genes

A heterologous nucleotide sequence encoding an antigenic determinant ofa filovirus protein can be expressed as a single transcriptional unit.For example, a heterologous nucleotide sequence encoding an antigenicdeterminant of a filovirus protein can be operably linked to a poxviruse.g., vaccinia virus promoter and/or linked to a poxvirus e.g., vacciniavirus transcriptional terminator.

In certain embodiments, the “transcriptional unit” is inserted by itselfinto an insertion site in the MVA/FPV genome. In certain embodiments,the “transcriptional unit” is inserted with other transcriptionalunit(s) into an insertion site in the MVA/FPV genome. The“transcriptional unit” is not naturally occurring (i.e., it isheterologous, exogenous or foreign) in the MVA/FPV genome and is capableof transcription in infected cells.

Preferably, the recombinant MVA/FPV comprises 1, 2, 3, 4, 5, or moretranscriptional units inserted into the MVA/FPV genome. In certainembodiments, the recombinant MVA/FPV stably expresses heterologousnucleotide sequences encoding antigenic determinants of a filovirusprotein encoded by 1, 2, 3, 4, 5, or more transcriptional units. Incertain embodiments, the recombinant MVA/FPV comprises 2, 3, 4, 5, ormore transcriptional units inserted into the MVA/FPV genome at 1, 2, 3,or more insertion sites in the MVA/FPV genome.

In certain embodiments, expression of one, more, or all of theheterologous nucleotide sequences encoding antigenic determinants of afilovirus protein is under the control of one or more poxviruspromoters. In certain embodiments, the poxvirus promoter is a Pr7.5promoter, a hybrid early/late promoter, a PrS promoter, a PrS5Epromoter, a synthetic or natural early or late promoter, or a cowpoxvirus ATI promoter. Suitable promoters are further described in WO2010/060632, WO 2010/102822, WO 2013/189611 and WO 2014/063832incorporated fully by reference herewith. In certain embodiments, thepoxvirus promoter is selected from the group consisting of the PrSpromoter (SEQ ID NO:23), the PrS5E promoter (SEQ ID NO:24), the Pr7.5(SEQ ID NO:25), the PrLE1 promoter (SEQ ID NO:27), the Pr13.5 longpromoter (SEQ ID NO:35) and the FPV-40K promoter (SEQ ID NO:26), morepreferably selected from the group consisting of the PrS promoter (SEQID NO:23), the PrS5E promoter (SEQ ID NO:24), the Pr7.5 (SEQ ID NO:25)and the PrLE1 promoter (SEQ ID NO:27).

In certain embodiments, the nucleotide sequence encoding the antigenicdeterminant of the filovirus protein preferably the ZEBOV, SEBOV,EBOV-Cdl, MARV and NP-ZEBOV protein, more preferably theGP-ZEBOV-Mayinga, GP-SEBOV-Gulu, GP-MARV and NP-ZEBOV, most preferablythe GP-MARV-Musoke or GP-MARV-Angola are under the control of thepromoter selected from the group consisting of PrS, PrLE1 and Pr7.5. Ina preferred embodiment, the nucleotide sequence encoding the antigenicdeterminant of the filovirus protein GP-SEBOV and GP-MARV-Musoke areexpressed under the control of the PrS promoter (e.g., SEQ ID NO:23),NP-EBOV-Cdl is expressed under the control of the PrLE1 or modifiedPrLE1 promoter (e.g., SEQ ID NO:27 and SEQ ID NO:32), andGP-ZEBOV-Mayinga is expressed under the control of the Pr7.5 promoter(e.g., SEQ ID NO:25).

In another preferred embodiment, the nucleotide sequence encoding theantigenic determinant of the filovirus protein of the FPV of any of theembodiments is under the control of the promoter, preferably includingor having SEQ ID NO:26.

Filovirus Vaccines and Pharmaceutical Compositions

Since the recombinant MVA viruses described herein are highlyreplication restricted and, thus, highly attenuated, they are idealcandidates for the treatment of a wide range of mammals including humansand even immune-compromised humans. Hence, provided herein arepharmaceutical compositions and vaccines for inducing an immune responsein a living animal body, including a human. Additionally provided is arecombinant MVA vector comprising a nucleotide sequence encoding anantigenic determinant of a filovirus glycoprotein for use in thetreatment and/or prevention of a filovirus-caused disease.

The vaccine preferably comprises any of the recombinant MVA virusesdescribed herein formulated in solution in a concentration range of 10⁴to 10⁹ TCID₅₀/ml, 10⁵ to 5×10⁸ TCID₅₀/ml, 10⁶ to 10⁸ TCID₅₀/ml, or 10⁷to 10⁸ TCID₅₀/ml. A preferred vaccination dose for humans comprisesbetween 10⁶ to 10⁹ TCID₅₀, including a dose of 10⁶ TCID₅₀, 10⁷ TCID₅₀,or 10⁸ TCID₅₀.

The pharmaceutical compositions provided herein may generally includeone or more pharmaceutically acceptable and/or approved carriers,additives, antibiotics, preservatives, adjuvants, diluents and/orstabilizers. Such auxiliary substances can be water, saline, glycerol,ethanol, wetting or emulsifying agents, pH buffering substances, or thelike. Suitable carriers are typically large, slowly metabolizedmolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, lipidaggregates, or the like.

For the preparation of vaccines, the recombinant MVA viruses providedherein can be converted into a physiologically acceptable form. This canbe done based on experience in the preparation of poxvirus vaccines usedfor vaccination against smallpox as described by H. Stickl et al.,Dtsch. med. Wschr. 99:2386-2392 (1974).

For example, purified viruses can be stored at −80° C. with a titer of5×10⁸ TCID₅₀/ml formulated in about 10 mM Tris, 140 mM NaCl pH 7.4. Forthe preparation of vaccine shots, e.g., 10²-10⁸ or 10²-10⁹ particles ofthe virus can be lyophilized in 100 ml of phosphate-buffered saline(PBS) in the presence of 2% peptone and 1% human albumin in an ampoule,preferably a glass ampoule. Alternatively, the vaccine shots can beproduced by stepwise freeze-drying of the virus in a formulation. Thisformulation can contain additional additives such as mannitol, dextran,sugar, glycine, lactose or polyvinylpyrrolidone or other aids such asantioxidants or inert gas, stabilizers or recombinant proteins (e.g.,human serum albumin) suitable for in vivo administration. The glassampoule is then sealed and can be stored between 4° C. and roomtemperature for several months. However, as long as no need exists, theampoule is stored preferably at temperatures below −20° C.

For vaccination or therapy, the lyophilisate can be dissolved in anaqueous solution, preferably physiological saline or Tris buffer, andadministered either systemically or locally, i.e., parenteral,subcutaneous, intravenous, intramuscular, intranasal, or any other pathof administration known to the skilled practitioner. The mode ofadministration, the dose and the number of administrations can beoptimized by those skilled in the art in a known manner. However, mostcommonly a patient is vaccinated with a second shot about one month tosix weeks after the first vaccination shot.

Combination Vaccines Using Homologous/Heterologous Prime-Boost Regimens

The Combination Vaccines and methods described herein may also be usedas part of a homologous prime-boost regimen. In the homologousprime-boost, a first priming vaccination is given followed by one ormore subsequent boosting vaccinations. The boosting vaccinations areconfigured to boost the immune response generated in the firstvaccination by administration of the same recombinant poxvirus that wasused in the first vaccination.

In one exemplary embodiment a homologous prime-boost regimen may beemployed wherein a MVA viral vector as defined herein is administered ina first dosage. One or more subsequent administrations of a MVA viralvector as defined herein can be given to boost the immune responseprovided in the first administration. Preferably, the one or moreantigenic determinants are the same or similar to those of the firstadministration.

The MVA and FPV recombinant viral vectors according to the presentinvention may also be used in heterologous prime-boost regimens in whichone or more of the initial prime vaccinations are done with either theMVA or the FPV vector as defined herein and one or more subsequentboosting vaccinations are done with the poxviral vector not used in theprime vaccination, e.g., if a MVA vector defined herein is given in aprime boost, then subsequent boosting vaccinations would be FPV vectorsand vice versa.

In a preferred embodiment the prime vaccination is done with the MVAvector and the boosting vaccination with the FPV. Accordingly, oneaspect of the invention relates to a combination vaccine comprising:

-   -   a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of at least one filovirus subtype, together        with a pharmaceutically acceptable carrier; and    -   b) a second composition comprising an immunologically effective        amount of a fowlpox vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        wherein the first compositions is a priming composition and the        second composition is a boosting composition, preferably wherein        the boosting composition comprises two or more doses of the        vector of the boosting composition.        Vaccines and Kits Comprising Recombinant MVA and FPV Viruses

Also provided herein are vaccines and kits comprising any one or more ofthe recombinant FPVs and/or MVAs described herein. The kit can compriseone or multiple containers or vials of the recombinant MVA or FPV,together with instructions for the administration of the recombinant MVAand FPV to a subject at risk of filovirus infection. In certainembodiments, the subject is a human. In certain embodiments, theinstructions indicate that the recombinant MVA is administered to thesubject in a single dose, or in multiple (i.e., 2, 3, 4, etc.) doses. Incertain embodiments, the instructions indicate that the recombinant MVAor FPV virus is administered in a first (priming) and second (boosting)administration to naïve or non-naïve subjects. Preferably, a kitcomprises at least two vials for prime/boost immunization comprising therecombinant MVAs as described herein for a first inoculation (“priminginoculation”) in a first vial/container and for an at least secondand/or third and/or further inoculation (“boosting inoculation”) in asecond and/or further vial/container.

In a preferred embodiment the vaccines and kits provided herein comprisea first composition which comprises a MVA vector comprising a nucleicacid encoding an antigenic protein of a second filovirus subtype, of athird filovirus subtype or at least four filovirus subtypes.

In a preferred embodiment, the vaccines and kits provided hereincomprise a MVA vector in the first composition, which comprises anucleic acid encoding an antigenic protein selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 20, SEQID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:37.

In a further embodiment the vaccines and kits provided herein comprise aMVA vector in the first composition comprising a nucleic acid encodingan antigenic protein selected from the group having SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:29 and SEQ ID NO:31,preferably comprising a nucleic acid encoding an antigenic proteinselected from the group having: SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:29and SEQ ID NO:31.

In a further embodiment the vaccines and kits provided herein comprise afirst composition which comprises a MVA vector comprising a nucleic acidencoding an antigenic protein of at least four filovirus subtypes,preferably wherein the four different filovirus subtypes are selectedfrom the group having SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:37.

In a further preferred embodiment the vaccines and kits provided hereinare for use in generating a protective immune response against at leastone filovirus subtype, wherein the first composition is used for primingsaid immune response and the second composition is used for boostingsaid immune response or for use in generating a protective immuneresponse against at least one filovirus subtype, wherein the secondcomposition is used for priming said immune response and the firstcomposition is used for boosting said immune response. In any of thevaccines and kits provided herein the boosting composition can comprisetwo or more doses of the vector of the boosting composition.

As discussed previously above, the present invention also relates toheterologous vaccination regimes using two different non-replicatingviral vectors.

For heterologous vaccine programs, the present invention provides acombination vaccine and/or vaccination kit which comprises:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a fowlpox vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

The present invention also provides a combination vaccine and/orvaccination kit which comprises:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

In this embodiment, the combination vaccines and/or kit comprises atleast two vials for prime/boost immunization comprising the recombinantMVAs/FPVs as described herein for a first inoculation (“priminginoculation”) in a first vial/container and for an at least secondand/or third and/or further inoculation (“boosting inoculation”) in asecond and/or further vial/container.

The combination vaccine and/or kit can comprise multiple containers orvials of the recombinant MVA/FPV, together with instructions for theadministration of the recombinant MVA/FPV to a subject at risk offilovirus infection. In certain embodiments, the subject is a human. Incertain embodiments, the instructions indicate that the recombinantMVA/FPV is administered to the subject in a single dose, or in multiple(i.e., 2, 3, 4, etc.) doses. In certain embodiments, the instructionsindicate that the recombinant MVA/FPV virus is administered in a first(priming) and second (boosting) administration to naïve or non-naïvesubjects.

The first and/or second composition or MVA and/or FPV of any combinationvaccine, vaccination kit and/or any heterologous vaccine program of theinvention can comprise any of the MVA and/or FPV vector described hereinor as further defined under “Recombinant MVA and FPV” and anycombination thereof.

In a preferred embodiment, the combination vaccines as provided hereincomprise a first composition comprising a MVA vector comprising anucleic acid encoding an antigenic protein of a second filovirussubtype, an antigenic determinant of a third filovirus subtype, anantigenic determinant of four filovirus subtypes or an antigenicdeterminant of at least four filovirus subtypes.

In another embodiment, the combination vaccines as provided hereincomprise a filovirus subtype selected from an Ebola virus (EBOV) or aMarburg virus (MARV).

In another embodiment, the combination vaccines as provided hereincomprises an antigenic determinant from one or more EBOV subtypesselected from the group consisting of Zaire Ebola virus (ZEBOV), SudanEbola virus (SEBOV), Cote d'Ivoire Ebola virus (EBOV-Cdl), Reston Ebolavirus (EBOV-Reston) and Bundibugyo Ebola virus (BEBOV).

In another embodiment, the combination vaccines as provided hereincomprise an antigenic determinant of the filovirus protein is selectedfrom the group consisting of an envelope glycoprotein (GP),nucleoprotein (NP), virion protein 35 (VP35), virion protein 40 (VP40),virion protein 30 (VP30), virion protein 24 (VP24), and RNA-directed RNApolymerase protein (L).

In a further preferred embodiment, the combination vaccines as providedherein comprise a MVA vector in the first composition comprising anucleic acid encoding an antigenic protein selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 20, SEQID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:37.

In a further embodiment, the combination vaccines provided hereincomprise a first composition which comprises a MVA vector comprising anucleic acid encoding an antigenic protein of at least four filovirussubtypes, preferably wherein the four different filovirus subtypes areselected from the group having SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO: 20, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ IDNO:37.

In a further preferred embodiment, the combination vaccines providedherein comprise a first composition which comprises a MVA vectorcomprising a nucleic acid encoding antigenic proteins from fourdifferent filovirus subtypes selected from the group having: SEQ IDNO:6, SEQ ID NO:20, SEQ ID NO:29 and SEQ ID NO:31.

In another embodiment, the combination vaccines provided herein are foruse in generating a protective immune response against at least onefilovirus subtype, preferably at least two, more preferably at leastfour filovirus subtype.

In another embodiment of the present invention, the present inventionrelates to a combination vaccine or the recombinant MVA of any of theembodiments for use as a medicament or vaccine for generating aprotective immune response or for inducing an enhanced immune responseagainst at least one filovirus subtype, at least two filovirus subtypes,at least three or at least four filovirus subtypes, wherein the MVA iscapable of producing filovirus-like particles in the subject to betreated, preferably, wherein the MVA is producing filovirus-likeparticles in the subject to be treated.

Methods and Uses of Recombinant MVA/FPV Viruses

Also provided herein are methods and/or any of the recombinant MVAs/FPVsas described herein for use in a method of immunizing a subject animalor for affecting an immune response in a subject. Also covered are usesof the recombinant MVAs/FPVs described herein for the preparation of amedicament or pharmaceutical for the immunization of a subject animal,in particular for the preparation of a medicament or vaccine fortreating and/or preventing a filovirus-caused disease in a subject.Provided are also recombinant MVA/FPV according to any embodiment hereinfor use in priming or boosting an immune response against a filovirus,preferably wherein the recombinant MVA and/or recombinant FPV isadministered once, twice, three times or four times.

Further covered herein are vaccine combinations or recombinant MVA ofany of the embodiments for use as a medicament or vaccine for inducingan enhanced immune response against a filovirus infection wherein theMVA is capable of producing filovirus-like particles in the subject tobe treated, preferably, wherein the MVA is producing filovirus-likeparticles in the subject to be treated. Also covered are vaccinecombinations or recombinant MVA of any of the embodiments for use as amedicament or vaccine for treating and/or preventing a filovirusdisease, wherein the MVA is capable of producing filovirus-likeparticles in the subject to be treated, preferably, wherein the MVA isproducing filovirus-like particles in the subject to be treated.

Accordingly, in one embodiment, the present invention provides a methodof inducing an immune response against filovirus in a subject, themethod comprising administering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a fowlpox vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

In another embodiment, the invention provides a method of inducing animmune response against a filovirus in a subject, the method comprisingadministering to the subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes, together        with a pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        wherein one of the compositions is a priming composition and the        other composition is a boosting composition.

In another embodiment, the method of inducing an immune response againsta filovirus, the uses of the recombinant MVAs/FPVs described herein forthe preparation of a medicament for immunization of a subject animal, inparticular for the preparation of a medicament or vaccine for treatingand/or preventing a filovirus-caused disease in a subject or thecombination vaccine of any of the embodiments for use of providing aprotective immune response against a filovirus infection as providedherein comprises a first composition which comprises a MVA vectorcomprising a nucleic acid encoding an antigenic protein of a secondfilovirus subtype, of a third filovirus subtype or of at least fourfilovirus subtypes.

In another embodiment, the method of inducing an immune response againsta filovirus, the uses of the recombinant MVAs/FPVs described herein forthe preparation of a medicament for immunization of a subject animal, inparticular for the preparation of a medicament or vaccine for treatingand/or preventing a filovirus-caused disease in a subject or thecombination vaccine of any of the embodiments for use of providing aprotective immune response against a filovirus infection as providedherein comprise a MVA vector in the first composition which comprises anucleic acid encoding an antigenic protein selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 20, SEQID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:37.

In a further embodiment, the method of inducing an immune responseagainst a filovirus, the uses of the recombinant MVAs/FPVs describedherein for the preparation of a medicament for immunization of a subjectanimal, in particular for the preparation of a medicament or vaccine fortreating and/or preventing a filovirus-caused disease in a subject orthe combination vaccine of any of the embodiments for use of providing aprotective immune response against a filovirus infection as providedherein comprises a first composition which comprises a MVA vectorcomprising a nucleic acid encoding an antigenic protein of at least fourfilovirus subtypes, preferably wherein the four different filovirussubtypes are selected from the group having SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO: 20, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34 andSEQ ID NO:37.

In another embodiment, the present invention provides a method ofproviding protective immunity and/or a protective immune responseagainst a filovirus infection in a subject. In another embodiment, theinvention provides a method of providing protective immunity and/or aprotective immune response against a filovirus infection in a subject:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding        antigenic proteins of at least two filovirus subtypes,        preferably at least three or at least four different filovirus        subtypes, together with a pharmaceutically acceptable carrier;        and    -   (b) a second composition comprising an immunologically effective        amount of a FPV vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype, together with a        pharmaceutically acceptable carrier;        wherein one of the compositions is a priming composition and the        other composition is a boosting composition, preferably wherein        the second composition is a boosting composition, preferably to        be administered once, twice, three times or four times.

In another embodiment, the method of providing protective immunityand/or a protective immune response against a filovirus infection of anyof the embodiments comprises a MVA vector in the first compositioncomprising a nucleic acid encoding an antigenic protein selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO: 20, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ ID NO:37.

In another embodiment, the method of providing protective immunityand/or a protective immune response against a filovirus infection of anyof the embodiments comprises a MVA vector in the first compositioncomprising a nucleic acid encoding antigenic proteins from fourdifferent filovirus subtypes having SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO: 20, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34 and SEQ IDNO:37.

In another embodiment, the present invention provides a method forproduction of filovirus-like particles or a method of inducing anenhanced immune response against a filovirus in a subject, the methodcomprising production of filovirus-like particles in the subject of anyof the embodiments, wherein the filovirus VP40 is selected from thegroup consisting of Zaire Ebola virus (ZEBOV), Sudan Ebola virus(SEBOV), Cote d'Ivoire Ebola virus (EBOV-Cdl), Reston Ebola virus(EBOV-Reston) and Bundibugyo Ebola virus (BEBOV), preferably wherein thefilovirus VP40 is selected from one or more ZEBOV, SEBOV and MARV.

In another embodiment, the present invention provides a method forproduction of filovirus-like particles or a method of inducing anenhanced immune response against a filovirus in a subject, the methodcomprising production of filovirus-like particles in the subject of anyof the embodiments, wherein the filovirus glycoprotein and the filovirusVP40 are selected from the same filovirus strain.

In another embodiment, the present invention provides a method forproduction of filovirus-like particles or a method of inducing anenhanced immune response against a filovirus in a subject, the methodcomprising production of filovirus-like particles in the subject of anyof the embodiments, wherein the MVA vector further comprises a nucleicacid encoding a filovirus nucleoprotein (NP), preferably wherein thefilovirus nucleoprotein and the filovirus VP40 are derived from the samefilovirus strain.

In another embodiment, the filovirus strain of any of the above methodsis selected from the group of Zaire-Mayinga, Zaire-Kikwit, Zaire-Gabon,Cote d'Ivoire Ebola virus, Sudan-Boniface, Sudan-Maleo, Sudan-Gulu,Marburg-Ravn, Marburg-Ozolin, Marburg-Ratayczak, Marburg-Musoke,Marburg-Angola, preferably Zaire-Mayinga or Cote d'Ivoire Ebola virus,preferably wherein the filovirus VP40 is selected from the group ofZaire-Mayinga or Marburg-Musoke, more preferably wherein the filovirusVP40 comprises a nucleic acid encoding the protein sequence of SEQ IDNO:34 or wherein the nucleic acid encoding the antigenic protein of thefilovirus VP40 comprises SEQ ID NO:33.

As used herein, the term “protective immunity” or “protective immuneresponse” means that the vaccinated subject is able to control aninfection with the pathogenic agent against which the vaccination wasdone. Usually, the subject having developed a “protective immuneresponse” develops only mild to moderate clinical symptoms or nosymptoms at all. Usually, a subject having a “protective immuneresponse” or “protective immunity” against a certain agent will not dieas a result of the infection with said agent. In certain embodiments,the subject animal is a mammal. The mammal may be an adult cow, a calf,in particular a juvenile calf, a rat, rabbit, pig, mouse, but preferablya human, and the method comprises administering a dose of any one ormore of the recombinant MVAs/FPVs provided herein to the subject.

In certain embodiments, the subject is a human. In certain embodiments,the subject is an adult. In certain embodiments, the adult isimmune-compromised. In certain embodiments, the adult is over the age of10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years.In certain embodiments, the subject's age is less than 5 years, lessthan 3 years, less than 2 years, less than 15 months, less than 12months, less than 9 months, less than 6, or less than 3 months. Incertain embodiments, the subject's age is from 0-3 months, 3-6 months,6-9 months, 9-12 months, 1-2 years, or 2-5 years.

Any of the recombinant MVAs/FPVs provided herein may be administered tothe subject at a dose of 10⁶ to 10¹⁰ TCID₅₀, preferably 10⁸ to 10⁹TCID₅₀ as, e.g., at a dose of 10⁶ to 10⁹ TCID₅₀, 10⁶ to 5×10⁸ TCID₅₀,10⁷ to 10⁸ TCID₅₀, 5×10⁷ TCID₅₀ to 5×10⁸ TCID₅₀, 10⁷ TCID₅₀ or 10⁸TCID₅₀. In a certain embodiment, the recombinant MVA/FPV vector isadministered in an amount of 1×10⁸ TCID₅₀ to 1×10¹⁰ TCID₅₀. In anotherembodiment, the recombinant MVA/FPV is administered in an amount of1×10⁸ TCID₅₀ to 5×10⁹, preferably in an amount of 5×10⁸ TCID₅₀ to 6×10⁹.In certain embodiments, any of the recombinant MVAs provided herein areadministered to a human subject at a dose of 10⁷ TCID₅₀ or 10⁸ TCID₅₀ or5×10⁸ TCID₅₀. In certain embodiments, any of the recombinant FPVsprovided herein is administered to a human subject at a dose of 5×10⁸,6.3×10⁸ or 1×10⁹ TCID₅₀.

In another embodiment, the recombinant MVAs provided herein areadministered to a human subject at a dose lower than the recombinantFPVs. In certain embodiments, any of the recombinant MVAs/FPVs providedherein are administered to the subject at any of the doses providedherein prior to filovirus exposure as, e.g., 1, 2, 3, or 4 weeks or 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before filovirus exposure.In certain embodiments, any of the recombinant MVAs/FPVs provided hereinis administered to the subject at any of the doses provided herein afterfilovirus exposure as, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or 1, 2, 3, 4, 5, 6,or 7 days after filovirus exposure.

In certain embodiments, the recombinant MVAs/FPVs provided herein areadministered to the subject in a single dose, or in multiple (i.e., 2,3, 4, etc.) doses. In certain embodiments, the recombinant MVAs/FPVsprovided herein are administered in a first (priming) and second(boosting) administration. The first dose may comprise 10⁷ to 10⁸ TCID₅₀of recombinant MVA/FPV virus and the second dose may comprise 10⁷ to 10⁸TCID₅₀ of recombinant MVA/FPV virus.

Boosting compositions are generally administered once or multiple timesweeks or months after administration of the priming composition, forexample, about 1 or 2 weeks or 3 weeks, or 4 weeks, or 6 weeks, or 8weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeksor one to two years.

Preferably, the initial boosting inoculation is administered 1-12 weeksor 2-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks afterpriming. In a preferred embodiment, the initial boosting inoculation isadministered 4 or 8 weeks after priming. In additional preferredembodiments, the initial boosting is conducted at least 2 weeks or atleast 4 weeks after priming. In still another preferred embodiment, theinitial boosting is conducted 4-12 weeks or 4-8 weeks after priming.

The recombinant MVAs/FPVs provided herein can be administeredsystemically or locally. In certain embodiments, the recombinantMVAs/FPVs are administered parenterally, subcutaneously, intravenously,intramuscularly, or intranasally, in particular subcutaneously.Preferably, the recombinant MVAs/FPVs are administered intranasally. Inother embodiments, the recombinant MVAs/FPVs are administered by anyother path of administration known to the skilled practitioner. In afurther preferred embodiment, the recombinant MVA/FPV is administeredintramuscularly, preferably the recombinant MVA/FPV is administeredintramuscularly in a volume ranging between about 100 μl to about 10 mlpreferably containing concentrations of e.g., about 10⁴ to 10¹⁰ virusparticles/ml. Preferably, the recombinant MVA/FPV vector is administeredin a volume ranging between 0.25 and 1.0 ml. More preferably, therecombinant MVA/FPV vector is administered in a volume of about 0.5 ml.

Method for Producing a Recombinant MVA/FPV Vector

Further embodiments comprise a method for producing a recombinant MVAvector of any of the embodiments of the invention or the antigenicdeterminant expressed from the genome of said recombinant MVA vector,comprising the steps of

-   -   (a) infecting a host cell with the recombinant MVA virus of any        of the embodiments or transfecting the cell with the recombinant        DNA of the recombinant MVA virus of any of the embodiments        preferably with the addition of a helper virus for production of        MVA virus particles,    -   (b) cultivating the infected or transfected cell, and    -   (c) isolating the MVA virus and/or the antigenic determinant        from said cell.

In another embodiment, the invention relates to a recombinant MVA virusand/or an antigenic determinant obtained from the method for producing arecombinant vector.

Further embodiments comprise a method for producing a recombinant FPVvector of any of the embodiments of the invention or the antigenicdeterminant expressed from the genome of said recombinant FPV vector,comprising the steps of

-   -   (a) infecting a host cell with the recombinant FPV virus of any        of the embodiments or transfecting the cell with the recombinant        DNA of the recombinant FPV virus of any of the embodiments        preferably with the addition of a helper virus for production of        FPV virus particles,    -   (b) cultivating the infected or transfected cell, and    -   (c) isolating the FPV virus and/or the antigenic determinant        from said cell.

In another embodiment, the invention relates to a recombinant FPV virusand/or an antigenic determinant obtained from the method for producing arecombinant vector.

In another embodiment, the invention relates to a method of generating arecombinant MVA vector comprising the steps of:

-   -   (a) infecting a host cell with a MVA virus,    -   (b) transfecting the infected cell with a recombinant vector        comprising at least one nucleotide sequence encoding an        antigenic determinant of any of the proteins, said nucleic acid        sequence further comprising a genomic MVA virus sequence capable        of directing the integration of the at least one nucleotide        sequence into the MVA virus genome, and    -   (c) identifying, isolating and optionally purifying the        generated recombinant MVA virus.

In another embodiment, the invention relates to a method of generating arecombinant FPV vector comprising the steps of:

-   -   (a) infecting a host cell with an FPV virus,    -   (b) transfecting the infected cell with a recombinant vector        comprising at least one nucleotide sequence encoding an        antigenic determinant of any of the proteins, said nucleic acid        sequence further comprising a genomic FPV virus sequence capable        of directing the integration of the at least one nucleotide        sequence into the FPV virus genome, and    -   (c) identifying, isolating and optionally purifying the        generated recombinant FPV virus.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the appended claims.

EXAMPLES

The detailed examples which follow are intended to contribute to abetter understanding of the present invention. However, the invention isnot limited by the examples. Other embodiments of the invention will beapparent to those skilled in the art from consideration of thespecification and practice of the invention disclosed herein.

Example 1: Construction of Recombinant MVA

The following sections describe construction of recombinant MVAscomprising one or more heterologous nucleic acids expressing anantigenic determinant of a filovirus envelop glycoprotein and/or afurther filovirus protein. All other constructs described herein aremade using similar methods.

Construction of MVA-mBN252B (PrS-GP-MARV-Musoke)

The full-length DNA sequence of the naturally-occurring GP-MARV-Musokegene (Lake Victoria isolate) served as reference sequence forconstruction of MARV vaccine candidate MVA-mBN252B. A nucleotidesequence encoding full-length GP-MARV-Musoke was synthesized by GeneartAG (Regensburg, Germany) with codon usage optimized for expression inhumans and to minimize or prevent internal homologous recombinationevents. Although the codon optimization changed the wild-type DNAsequence, the codon-optimized sequence encodes an amino acid sequenceidentical to the wild-type GP-MARV-Musoke (SEQ ID NO:6; NCBI accessionnumber ABA87127.1). Expression of GP-MARV-Musoke is driven by thepromoter PrS, a synthetic promoter designed from early and late elementsof vaccinia virus promoters (SEQ ID NO:23; see also S. Chakrabarti etal., “Compact, Synthetic Vaccinia Virus Early/Late Promoter for ProteinExpression”, BioTechniques 23(6):1094-1097 (1997)). The codon-optimizedGP-MARV-Musoke gene was inserted into the MVA-BN genome by standardmethods (see below) using one of several customized recombinationplasmids targeting different specific regions of the MVA-BN genome,including the deletion sites or the intergenic (non-coding) regions(IGR).

To insert the codon-optimized GP-MARV-Musoke gene into the MVA-BNgenome, chicken embryonic fibroblast cells (CEF cells) were infectedwith MVA-BN and subsequently transfected with the recombination plasmidpBN433 (FIG. 5A). pBN433 contained the codon-optimized GP-MARV-Musokegene (SEQ ID NO:5 (DNA) encoding SEQ ID NO:6 (amino acid)) under controlof the synthetic PrS promoter inserted via BspEI/NheI restriction intoplasmid pBNX197 (FIG. 4B). Plasmid pBN433 also contains MVA-BN DNAsequences flanking IGR 148/149 in the MVA-BN genome and a selectioncassette flanked by loxP sites, which allows later elimination of theselection cassette by Cre recombinase-mediated recombination. Followinghomologous recombination between flanking sequences in the plasmid andhomologous sequences at the desired insertion site in the MVA-BN genome(i.e., IGR 148/149), the coding portion of the plasmid was inserted intothe desired site in the MVA-BN genome.

After amplification and plaque purification (nine passages; three ofthem including plaque purification) under selective conditions(mycophenolic acid/xanthine and hypoxanthine), the recombinant MVA-BNproduct designated MVA-mBN252A (PreMaster A), containing the gene forGP-MARV-Musoke was obtained. Recombinant MVA-mBN252A PreMaster virusstock was examined for elimination of MVA-BN (parental virus; data notshown), for correct sequence of the inserted gene together with theinsertion flanking regions (by gene-specific PCR using primers specificfor the MVA-BN genomic sequence into which the foreign gene wasinserted; data not shown), for absence of microbes (sterility test; datanot shown), and for the presence and correct size of the insert (bysequencing; data not shown). The titer of the MVA-mBN252A PreMastervirus stock was also determined.

The presence of a selection cassette in the inserted sequence permitspositive selection for recombinant MVA-BN viruses in culture. Togenerate the final recombinant MVA-mBN252B, the selection cassette wasremoved from MVA-mBN252A PreMaster virus stock using the Cre/loxPsystem. To remove the selection cassette, CEF cells infected withrecombinant MVA-BN containing the insert of plasmid pBN433 (i.e.,GP-MARV-Musoke under the control of the PrS promoter, plus a selectioncassette flanked by loxP sites) were further transfected with pBN274, anexpression plasmid encoding the CRE recombinase (FIG. 4C). Thesite-specific Cre-recombinase catalyzed the precise excision of theselection cassette DNA sequences flanked by the target loxP sequence,completely removing the selection cassette. The resulting virus wasplaque purified under non-selective conditions (twenty seven passages;nine of them including plaque purification), and the recombinant virusMVA-mBN252B devoid of selection cassette was isolated. Completeelimination of the selection cassette was confirmed by nested PCR (datanot shown). Finally, expression of GP-MARV-Musoke by recombinantMVA-mBN252B was confirmed by reverse-transcriptase PCR (RT-PCR; data notshown).

Construction of MVA-mBN226B (Multi-Antigen MVA-Filo)

For all transgenes expressed from MVA-mBN226B, the full-length DNAsequences of the naturally-occurring genes served as referencesequences. Those were synthesized by Geneart AG (Regensburg, Germany)with codon usage optimized for expression in humans and to minimize orprevent internal homologous recombination events. The codon optimizationchanged the wild-type DNA sequence without altering the amino acidsequence. MVA-mBN226B contains the following filoviral genes: GP-SEBOV(SEQ ID NO:30); NP-EBOV-Cdl (SEQ ID NO:28); GP-ZEBOV, Mayinga strain(GP-ZEBOV-Mayinga, SEQ ID NO:19) and GP-MARV-Musoke (SEQ ID NO:5).GP-SEBOV and GP-MARV-Musoke are expressed under the control of the PrSpromoter (SEQ ID NO:23), NP-EBOV-Cdl is expressed under the control ofthe PrLE1 or modified PrLE1 promoter (SEQ ID NO:27 and SEQ ID NO:32),and GP-ZEBOV-Mayinga is expressed under the control of the Pr7.5promoter (SEQ ID NO:25).

The PrS promoter is a synthetic promoter designed from early and lateelements of Vaccinia virus promoters, which ensures transgene expressionduring both the early and late phases of gene expression. Similarly, thePr7.5 promoter from the vaccinia virus 7.5 kDa gene is a strong earlyand late promoter, meaning transgenes under its control will also beexpressed during both the early and late phases of gene expression (SEQID NO:25; see also M. A. Cochran et al., “In vitro mutagenesis of thepromoter region for a vaccinia virus gene: evidence for tandem early andlate regulatory signals”, J. Virol. 54(1):30-37 (1985)). The promoterPrLE1 is a synthetic promoter consisting of the A-type inclusion bodypromoter of cowpox virus (ATI) fused to five optimized early elementsderived from Pr7.5 (SEQ ID NO:27; see also K. Baur et al.,“Immediate-Early Expression of a Recombinant Antigen by ModifiedVaccinia Virus Ankara Breaks the Immunodominance of StrongVector-Specific B8R Antigen in Acute and Memory CD8 T-Cell Responses”,J. Virol. 84(17):8743-8752 (2010)). Consequently, NP-EBOV-Cdl will beexpressed during both the early and late phases of expression. Moreover,PrLE1 was shown to induce especially strong cell-mediated immuneresponses. During passaging of MVA-mBN226B, one of the five earlyelements derived from Pr7.5 was lost, likely by homologousrecombination; analysis showed sufficient expression levels ofNP-EBOV-Cdl, however (data not shown), so the modified construct wasused without replacing the modified PrLE1 promoter.

For the insertion of foreign genes into the MVA-BN genome severalrecombination plasmids that target the different deletions andintergenic regions (IGR) of the MVA-BN genome were generated. Togenerate recombinant MVA-BN products, foreign sequences of interest canbe inserted into any of these basic vectors, e.g., pBNX186 targeting IGR88/89 (see FIG. 4A) or pBNX197 targeting IGR 148/149 (see FIG. 4B),using commonly available restriction enzymes and conventional molecularbiology techniques. To produce recombinant MVA-BN isolates expressingthe desired transgenes, CEF cells are then infected with MVA-BN andsubsequently transfected with one or more recombination plasmidsexpressing the desired transgene or transgenes and including a selectioncassette enabling positive selection for recombinant viruses. Duringhomologous recombination, the plasmid flanking sequences recombine withthe homologous sequences of the insertion site in the MVA-BN virusgenome. This inserts the plasmid sequences into the site targeted by thebasic vector used as starting material (e.g., IGR 148/149, IGR 88/89,etc.) in the MVA-BN genome. pBNX197 targets IGR 148/149 (FIG. 4B) andwas used as starting plasmid for construction of the final recombinationplasmid pBN384 (FIG. 5B). Plasmid pBN384 expresses GP-ZEBOV-Mayinga andGP-MARV-Musoke. pBNX 186 targets IGR 88/89 (FIG. 4A) and was used asstarting plasmid for construction of the final recombination plasmidpBN385 (FIG. 5C). Plasmid pBN385 expresses GP-SEBOV and NP-EBOV-Cdl.

To insert the GP-SEBOV, NP-EBOV-Cdl, GP-ZEBOV-Mayinga, andGP-MARV-Musoke transgenes into MVA-BN, CEF cells were infected withMVA-BN and subsequently transfected with the recombination plasmidspBN384 and pBN385. After amplification and plaque purification (tenpassages; three including plaque purification) under double selectiveconditions (mycophenolic acid/xanthine and hypoxanthine as well asGeneticin) the recombinant MVA-BN product designated MVA-mBN226A(Interim Premaster), containing the genes for three glycoproteins, onenucleoprotein and two selection cassettes, was obtained. RecombinantMVA-mBN226A PreMaster virus stock was examined for elimination of MVA-BN(parental virus; data not shown), for correct sequence of the insertedgenes together with the insertion flanking regions (by gene-specific PCRusing primers specific for the MVA-BN genomic sequences into which theforeign gene was inserted; data not shown), for absence of microbes(sterility test; data not shown), and for the presence and correct sizeof the inserts (by sequencing; data not shown). The titer of theMVA-mBN252A PreMaster virus stock was also determined.

After further amplification, removal of the selection cassettes andplaque purification under non-selective conditions (twenty passages; sixincluding plaque purification) recombinant virus MVA-mBN226B devoid ofselection cassettes was isolated. Complete elimination of the selectioncassettes was confirmed by nested PCR (data not shown). Finally,transgene expression by recombinant MVA-mBN226B was confirmed byreverse-transcriptase PCR (RT-PCR; data not shown).

Construction of MVA-mBN254A (MVA-GP-ZEBOV)

For the GP-ZEBOV transgene expressed from MVA-mBN254A, the full-lengthDNA sequence of the naturally-occurring gene served as referencesequences. The GP-ZEBOV gene was synthesized by Geneart AG (Regensburg,Germany) with codon usage optimized for expression in humans and tominimize or prevent internal homologous recombination events asdescribed above in “Construction of MVA-mBN226B”. The codon optimizationchanged the wild-type DNA sequence without altering the amino acidsequence. GP-ZEBOV-Mayinga is expressed under the control of the PrS5Epromoter (SEQ ID NO:24).

The PrS5E (SEQ ID NO:24) is a synthetic strong early and late promoterdesigned from the synthetic early and late promoter (Chakrabarti et al.,1997) followed by 5 early elements of the Pr7.5 promoter from thevaccinia virus 7.5 kDa gene (SEQ ID NO:25; see also M. A. Cochran etal., “In vitro mutagenesis of the promoter region for a vaccinia virusgene: evidence for tandem early and late regulatory signals”, J. Virol.54(1):30-37 (1985)). The PrS5E promoter is described in more detail inthe patent application WO 2013/189611A1.

For the insertion of foreign genes into the MVA-BN genome severalrecombination plasmids that target the different deletions andintergenic regions (IGR) of the MVA-BN genome were constructed. Togenerate recombinant MVA-BN products, foreign sequences of interest canbe inserted into any of these basic vectors, e.g., pBNX197 targeting IGR148/149 (see FIG. 4B), using commonly available restriction enzymes andconventional molecular biology techniques. To produce recombinant MVA-BNisolates expressing the desired transgenes, CEF cells are then infectedwith MVA-BN and subsequently transfected with one or more recombinationplasmids expressing the desired transgene or transgenes and including aselection cassette enabling positive selection for recombinant viruses.During homologous recombination, the plasmid flanking sequencesrecombine with the homologous sequences of the insertion site in theMVA-BN virus genome. This inserts the target sequences into the sitetargeted by the basic vector used as starting material (e.g., IGR148/149) in the MVA-BN genome. pBNX197 targets IGR 148/149 (FIG. 4B) andwas used as starting plasmid for construction of the final recombinationplasmid pBN436 (FIG. 5D). Plasmid pBN436 contains GP-ZEBOV-Mayinga.

To insert the GP-ZEBOV-Mayinga transgene into MVA-BN, CEF cells wereinfected with MVA-BN and subsequently transfected with the recombinationplasmid pBN436 (FIG. 5D). After amplification and plaque purification(nine passages; including three plaque purifications) under selectiveconditions (mycophenolic acid/xanthine and hypoxanthine) the recombinantMVA-BN product designated MVA-mBN254A (Premaster), containing the genefor GP-ZEBOV-Mayinga and the selection marker GPT-RFP fusion gene (FIG.3C). Recombinant MVA-mBN254A PreMaster virus stock was examined forelimination of MVA-BN (parental virus; data not shown), for correctsequence of the inserted genes together with the insertion flankingregions (by gene-specific PCR using primers specific for the MVA-BNgenomic sequences into which the foreign gene was inserted; data notshown), for absence of microbes (sterility test; data not shown), andfor the presence and correct size of the inserts (by sequencing; datanot shown). The titer of the MVA-mBN254A PreMaster virus stock was alsodetermined. Finally, transgene expression by recombinant MVA-mBN254A wasconfirmed by reverse-transcriptase PCR (RT-PCR; data not shown).

Other constructs were generated accordingly. In particular, MVA-mBN255expressed NP-EBOV-Cdl (SEQ ID NO:28) under the control of the PrSpromoter integrated into the IGR 88/99, VP40 ZEBOV (SEQ ID NO:33) undercontrol of the PrS promoter into the IGR 136/137 and GP-ZEBOV (SEQ IDNO:19) under control of the PrS5E promoter into the IGR 148/149 (FIG.13).

Construction of FPV-mBN368A (FPV-GP-ZEBOV) and FPV-mBN391(FPV-Multi-Filo)

For the GP-ZEBOV transgene expressed from FPV-mBN368A, the full-lengthDNA sequence of the naturally-occurring gene served as referencesequences. The GP-ZEBOV gene was synthesized by Geneart AG (Regensburg,Germany) with codon usage optimized for expression in humans asdescribed in “Construction of MVA-mBN226B”. The codon optimizationchanged the wild-type DNA sequence without altering the amino acidsequence. GP-ZEBOV-Mayinga is expressed under the control of the FPV-40Kpromoter (SEQ ID NO:26). The FPV-40K promoter is the FPV promotersequence of the 40K protein coding sequence in FPV.

For the insertion of foreign genes into the FPV genome, severalrecombination plasmids that target the different integration sites intothe FPV genome were constructed. To generate recombinant FPV products,foreign sequences of interest can be inserted into any of these basicvectors, e.g., pBNX221 targeting insertion site BamHI J (see FIG. 4D),using commonly available restriction enzymes and conventional molecularbiology techniques. To produce recombinant FPV isolates expressing thedesired transgenes, CEF cells are then infected with FPV andsubsequently transfected with one or more recombination plasmidsexpressing the desired transgene or transgenes and including a selectioncassette enabling positive selection for recombinant viruses. Duringhomologous recombination, the plasmid flanking sequences recombine withthe homologous sequences of the insertion site in the FPV virus genome.This inserts the target sequences into the site targeted by the basicvector used as starting material (e.g., insertion site BamHI J) in theFPV genome. pBNX221 targets insertion site BamHI J (FIG. 4D) and wasused as starting plasmid for construction of the final recombinationplasmid pBN555 (FIG. 5E). Plasmid pBN555 contains GP-ZEBOV-Mayinga undercontrol of the FPV-40K promoter.

To insert the GP-ZEBOV-Mayinga transgene into FPV, CEF cells wereinfected with FPV and subsequently transfected with the recombinationplasmid pBN555 (FIG. 5E). After amplification and plaque purification(13 passages; including four plaque purifications) under selectiveconditions (mycophenolic acid/xanthine and hypoxanthine) the recombinantMVA-BN product designated FPV-mBN368A (Premaster), containing the genefor GP-ZEBOV-Mayinga and the selection marker GPT-RFP fusion gene (FIG.3D), was obtained. Recombinant FPV-mBN368A PreMaster virus stock wasexamined for elimination of FPV (parental virus; data not shown), forcorrect sequence of the inserted genes together with the insertionflanking regions (by gene-specific PCR using primers specific for theFPV genomic flanking sequences into which the foreign gene was inserted;data not shown), for absence of microbes (sterility test; data notshown), and for the presence and correct size of the inserts (bysequencing; data not shown). The titer of the FPV-mBN368A PreMastervirus stock was also determined. Finally, transgene expression byrecombinant FPV-mBN368A was confirmed by reverse-transcriptase PCR(RT-PCR; data not shown).

Further fowlpox constructs were generated using FP14 (IGR 60/61) and theBamHI J region for integration according to the method as describedabove. FPV-mBN391 expressed GP-ZEBOV (SEQ ID NO:19 and 20) under thecontrol of the FPV-40K promoter (SEQ ID NO:26), GP-MARV-Musoke (SEQ IDNO:5 and 6) under the PrS promoter (SEQ ID NO:23) both at the FP14 siteand GP-MARV-Angola (SEQ ID NO:36 and 37) under the Pr13.5 long promoter(SEQ ID NO:35), GP-SEBOV (SEQ ID NO:30 and 31) under the FPV-40Kpromoter and NP-EBOV-Cdl (SEQ ID NO:28 and 29) under the control of thePrLE1 promoter (SEQ ID NO:27), all three inserted at the BamHI J regionin the order mentioned.

Example 2: MVA-BN-Filo (MVA-mBN226B) in Non-Human Primates

Immunogenicity and protective efficacy of MVA-BN-Filo (MVA-mBN226B) wasanalyzed in an Ebola and Marburg challenge model in cynomolgus macaques.Monkeys were housed and fed in accord with the appropriate institutionalguidelines for care and feeding of research animals.

The experimental design is set forth in Table 1 below.

TABLE 1 Vaccination protocol for MVA-BN-Filo in cynomolgus macaques.Challenge Virus Test/Reference Item Administration Administration GroupDose per Schedule Schedule Group Size Vaccination Admin. Route (Days)Virus (Day) 1 1 Vehicle — s.c. 0 and 28 EBOV 42 Control (TBS) 2 1Vehicle — s.c. 0 and 28 MARV 42 Control (TBS) 3 3 MVA-BN ®-Filo 5 × 10⁸s.c. 0 and 28 EBOV 42 TCID₅₀ 4 3 MVA-BN ®-Filo 5 × 10⁸ s.c. 0 and 28MARV 42 TCID₅₀ Intramuscular challenge (1,000 pfu) with either EBOVZaire strain or MARV Musoke strain; surviving animals were euthanized onDay 63

Dose volume was 0.5 mL for both vehicle control and vaccination groups;all vaccinations were delivered by subcutaneous injection. Firstvaccination day is designated Day 0. All animals received a challengedose of 1,000 pfu of ZEBOV (Groups 1 and 3) or MARV-Musoke (Groups 2 and4) via intramuscular injection on Day 42. All surviving animals wereeuthanized on Day 63.

GP-specific antibodies were measured by ELISA. As expected, thenon-vaccinated control animal challenged with MARV-Musoke had nodetectable GP-MARV-specific antibodies at any time prior to challenge(i.e., on Day 0, Day 28, and Day 36 (data not shown) and succumbed todisease. In contrast, two of the three animals vaccinated withMVA-BN-Filo (animal numbers 30766 and 30768) had low GP-MARV antibodytiters 28 days after the first vaccination (post first vaccination; seealso FIG. 6) and all three vaccinated animals showed a clear boostresponse eight days after the second vaccination (post boostervaccination; see FIG. 6). All three animals survived the otherwiselethal intramuscular challenge with MARV-Musoke.

Similarly, the non-vaccinated control animal challenged with ZEBOV wasnegative for GP-ZEBOV-specific antibodies at all time-points tested(data not shown). The control animal, as well as all vaccinated animalssuccumbed to infection following challenge with ZEBOV by intramuscularinjection. Surprisingly, all three vaccinated animals generatedGP-ZEBOV-specific antibodies prior to challenge, at levels greater inmagnitude than those measured in hyperimmune serum generated innon-human primates by vaccination with ZEBOV-GP. Complete necropsieswere performed on tissue collected in 10% neutral buffered formalin atthe time of death. Tissue sections were processed by routine methods,sectioned at 5 μm, and stained with hematoxylin and eosin forhistological evaluation. Findings are summarized in Table 2 below.

TABLE 2 Histological evaluation of vaccinated and control animals.Animal # 30763 30766 30765 30768 30764 30770 30769 30767 Necropsy #N11-05 N11-04 N11-06 N11-10 N11-07 N11-08 N11-09 N11-03 ChallengeMarburg Marburg Marburg Marburg Ebola Ebola Ebola Ebola Experimentalgroup control vaccine vaccine vaccine control control control controlSurvival 9 d€ 21 d 21 d 21 d 5 d (E) 7 d (SD) 6 d (E) 6 d(E) Liver:multifocal hepatic ++ − − − + ++ ++ − necrosis vasculitis +  ++* − ++* −− − − Lung: intra alveolar edema + − − − + + + − septal edema + − −− + + + − hemorrhage + − − − + + − − interstitial pneumonitis − − − − −+++ ++ − Spleen: hyperplasia − ++ +++ +++ − − − − lymphoid depletion ++− − − ++ +++ +++ ++ fibrin deposition red pulp +++ − − − ++ +++ + ++Splenic vasculitis +++ − − − ++ ++ + ++ Inguinal lymph node: macrophageinfiltration ++ − ++ − + + + +++ lymphoid depletion +++ − − − +++ +++ ++− Axillary lymph node: Macrophage infiltration − − − − − − − − lymphoiddepletion + − − − ++ ++ ++ + Mesenteric lymph node: Macrophageinfiltration − − − − − − − − lymphoid depletion ++ − − − ++ ++ ++ +Adrenal gland: necrosis − − − − +++ ++ − − *vasculitis in animals 30766and 30768 appears to be a pre-existing condition. E—euthanized;SD—spontaneous death

Analysis confirmed typical symptoms of hemorrhagic fever in theMARV-Musoke-challenged, non-vaccinated control animals, as well as inall ZEBOV-challenged animals, while vaccinated animals challenged withMARV-Musoke showed few histological changes, except for splenichyperplasia consistent with a post-challenge immune response and B-cellhyperplasia.

Results of the experiment are summarized in FIGS. 7A and 7B. FIG. 7Ashows that vaccination with MVA-BN-Filo protected 100% of animals fromchallenge with MARV-Musoke. FIG. 7B shows clinical scorespost-challenge; vaccinated animals challenged with MARV-Musoke showed nosymptoms or histological changes associated with hemorrhagic fever andharbored no virus in liver, spleen, adrenal glands, lymph nodes, orlungs.

Example 3: MVA-BN-Filo in Non-Human Primates

This experiment tested MVA-BN-Filo in non-human primates under a similarstudy protocol as described in Example 2 but challenged with anotherMarburg virus strain, i.e., Marburg Angola instead of Marburg Musoke inExample 2.

TABLE 3 Study design and outcome for MVA-BN-Filo in non-human primates.Test/Control Article Administration Group N Vaccination Dose ScheduleChallenge Survival 1 2 MVA-BN ® Filo 5 × 10⁸ 0, 28 Marburg 2/2 TCID₅₀Angola Day 56 2 3 MVA-BN ® MARV-IL15sushi 1 × 10⁸ 2/3 TCID₅₀ 3 3MVA-BN ®MARV-CD40L 1 × 10⁸ 3/3 TCID₅₀ 4 3 MVA-BN ® MARV-TRICOM 1 × 10⁸2/3 TCID₅₀ 5 1 TBS n/a 0/1 Intramuscular challenge (1,000 pfu) with MARVAngola strain; surviving animals were euthanized on Day 70.

Table 3 shows that MVA-BN-Filo completely protects non-human primatesagainst the Angola strain of Marburg virus. In contrast, thenon-vaccinated animal in group 5 succumbed to infection. It also showsthat protective efficacy is dose dependent, since a 5-fold lower doseusing MVA-BN-MARV, encoding also GP of Marburg virus, is only partiallyprotective, unless it co-expresses CD40L as co-stimulatory molecule. Allvaccine candidates produce antibodies specific for the MVA vector(Vaccinia specific antibodies), as well as antibodies specific for theMARV GP insert, as outlined in Table 4.

TABLE 4 Antibodies induced by MVA-BN-Filo in non-human primates. MVA-MVA- MVA- MVA- Multi- MARV MARV MARV Day 35 valent IL-15 CD40L TRICOMVaccinia-Elisa 21596 17838 6560 3246 Vaccinia-PRNT 1735 507 94 85MARV/GP-ELISA 298959 285187 199132 409941

Example 4: Heterologous Prime/Boost

This experiment tested the combination of recombinant MVA andrecombinant fowlpox FPV in prime/boost immunizations.

H-2K^(k+) B6CBA F1 mice (Janvier Labs, France) were immunizedsubcutaneously (s.c.) with 5×10⁷ TCID₅₀ MVA-ZEBOV-GP (MVA; MVA-mBN254A,FIG. 3C) or FPV-ZEBOV-GP (FPV; FPVmBN368A, FIG. 3D). The virus dose wasinjected at both flanks in a total volume of 100 μl/flank.

For the detection of ZEBOV-GP-specific IgG, 96-well plates (Corning, MA,USA) were coated with ZEBOV GP antigen (IBT Bioservices, MD, USA) at 4°C. over night. Duplicates of two-fold serum dilutions were added ontowashed and blocked plates and a sheep anti-mouse IgG-HRP (AbD Serotec,UK) was used as detection antibody. TMB substrate was added for 30minutes at RT and the reaction was stopped by the addition of 1M H₂SO₄.The absorbance was measured at 450 nm. The murine monoclonal antibody13F6 was used as a standard in order to calculate the serumconcentration of ZEBOV-GP IgG.

Mouse lymphocytes were freshly isolated from spleens by gently grindingand forcing the tissue through a 70 μm cell strainer (BD Bioscience, CA,USA). After erylysis, cells were incubated with 5 μg/ml ZEBOV-GP₅₇₇₋₅₈₄peptide (TELRTFSI) (SEQ ID NO:20) (GenScript, NJ, USA) for 6 hours at37° C. in complete RPMI in the presence of 10 μg/ml brefeldin A andCD107a-FITC. For live/dead discrimination, cells were stained using theZombie Aqua™ Fixable Viability kit (BioLegend, CA, USA). Intracellularstaining of IFN-′ and TNF-α was performed after surface staining withCD4-BV605, CD8α-BV421 (BioLegend, CA, USA) and CD44-APC-eFluor780(eBisocience, CA, USA) and fixation/permeabilization according to themanufacturers' instructions (BD Cytofix/Cytoperm, BD Biosciences). Allcells were acquired using a digital flow cytometer (LSR II, BDBiosciences, CA, USA) and data were analyzed with FlowJo software(FlowJo, OR, USA).

The four possible combinations of recombinant MVA and FPV prime/boostimmunizations were tested in H-2K^(k+) CBAB6 F1 mice, because a strongCD8 T cell epitope from Zaire Ebola virus (ZEBOV) glycoprotein (GP) wasdescribed for this MHC class I haplotype, namely GP₅₇₇₋₅₈₄ (TELRTFSI)(SEQ ID NO:20) (Rao et al., Vaccine 17(23-24):2991-8 (1999)). The serumconcentration of ZEBOV-GP-specific IgG was analyzed on day 21 and 41after s.c. immunization on day 0 and 21. While on day 21 allMVA-immunized mice had robust IgG titers, only 20% of FPV-immunized micehad seroconverted. After the second immunization, all animals wereseropositive for ZEBOV-GP-specific IgG. The lowest titers were observedafter homologous immunization with FPV. Between the animals immunizedtwice with MVA and those primed with FPV and boosted with MVA nodifference in the concentration of GP-specific IgG could be detected onday 41. The mice primed with MVA and boosted with FPV, however, hadslightly higher titers than all other groups on day 41 (FIG. 8A).

Interestingly, the same combination that resulted in the strongestantibody response also induced the strongest CD8 T cell response. Again,homologous immunization with FPV resulted in the weakest CTL response,followed by MVA-MVA and FPV-MVA immunizations. Priming with MVA followedby a boost with FPV induced ˜5-fold more cytotoxic CD8 T cells than thehomologous combination of 2×MVA (FIG. 8B).

Taken together, these data imply that heterologous immunization with MVAfirst and FPV second induces the strongest ZEBOV-GP-specific antibodyresponse and also the strongest CTL response, as shown by the presenceof highly functional CD8 T cells.

Example 5: Enhanced ZEBOV-GP Specific CD8 T Cell Response

H-2K^(k+) CBA mice were immunized s.c. with 5×10⁷ TCID₅₀ MVA or FPV onday 0 and 21. Mice (5 each per group) were sacrificed on day 42 for Tcell analysis by intracellular cytokine staining of splenocytes. Thefollowing prime/boost regimens were used: 1: MVA-ZEBOV-GP(mBN254)/FPV-ZEBOV-GP (mBN368), 2: MVA-multi-filo(MVA-mBN226)/FPV-ZEBOV-GP (mBN368), 3: MVA-ZEBOV-GP-VP40(mBN255)/FPV-ZEBOV-GP (mBN368). Data are summarized in FIG. 9.

Splenic CD8 T cell responses were analysed on day 42 after standard 6hour in vitro re-stimulation with 5 μg/ml ZEBOV-GP₅₇₇₋₅₈₄ peptide(TELRTFSI) (SEQ ID NO:20) in the presence of 10 μg/ml brefeldin A andanti-CD107a-FITC. Cells were surface stained with anti-CD4-BV605,anti-CD8-BV421, CD44-APC-eFluor780 and intracellularly withanti-IFN-γ-PECy7 and anti-TNF-α-PerCP-eFluor710. Live/deaddiscrimination was performed by LIVE/DEAD® Fixable Aqua Dead Cell StainKit according to the manufacturer's instruction (Life Technologies). Bargraphs show the total number of CD107a+, IFN-γ+ and TNF-α+CD8 T cells.Shown is the mean±SEM form 5 mice/group. The CD8 T cell response againstthe ZEBOV-GP-derived peptide TELFRTSI (SEQ ID NO: 20) was enhancedapproximately 2-fold when MVA-BN-ZEBOV/GP-VP40 was used as the primingconstruct in a MVA-FPV heterologous prime-boost regimen compared toMVA-ZEBOV-GP (mBN254) or MVA-multi-filo (MVA-mBN226) as primingconstructs (FIG. 9).

Example 6: Enhanced Protection of NHPs Against ZEBOV after Vaccinationwith MVA-GP-VP40

Cynomolgus macaques (Macaca fascicularis) were vaccinated twice (on Day0 and 28) subcutaneously with a dose of 5×10⁸ TCID₅₀ with eitherMVA-BN-ZEBOV/GP (n=3), MVA-BN-ZEBOV/GP-VP40 (n=3), or receivedTris-buffered saline (TBS) as negative control (placebo group, n=2).Prior to immunization and weekly before challenge (Days 7, 14, 21, 28,35 and 40) serum was collected for analysis by Ebola virus Zaireglycoprotein (GP)-specific and MVA-backbone-specific ELISA. Four weeksafter the booster vaccination, animals were challenged with Ebola virusZaire (Kikwit strain) by intramuscular administration of approximately1000 pfu.

TABLE 5 Study design: Vaccination Schedule Challenge Group Vaccine Dose(Route) Virus Schedule Survival 1 MVA-BN-ZEBOV/GP 5 × 10⁸ Day 0 + 28ZEBOV Kikwit 4 weeks 0/3 TCID₅₀ (s.c.) Approx. post last 1000 pfu i.m.vaccination 2 MVA-BN-ZEBOV/GP-VP40 5 × 10⁸ Day 0 + 28 2/3 TCID₅₀ (s.c.)3 TBS control n/a Day 0 + 28 0/2 (s.c.)Zaire Ebola Virus (ZEBOV)-Specific ELISA

An ELISA was performed determining ZEBOV/GP-specific antibodiesimmobilized by recombinant ZEBOV/GP and detected by a horse radishperoxidase (HRP)-conjugated antibody against NHP IgG. The amount ofbound HRP-labeled antibody was read out after a substrate reaction asoptical density (OD) value at 450 nm. The antibody concentration wascalculated according to the Four Parameter regression analysis and basedon a standard curve using monoclonal mouse antibody.

ELISA results are depicted in FIG. 10. All animals vaccinated with theMVA-BN-ZEBOV/GP or MVA-BN-ZEBOV/GP-VP40 construct had detectablebackbone- and ZEBOV-specific antibodies already after the primevaccination and antibody responses were boosted by a second vaccination.

In a second study cynomolgus macaques (Macaca fascicularis) werevaccinated three times (on Day 0, 28 and 56) subcutaneously with a doseof 5×10⁸ TCID₅₀ of either MVA-BN-multi-filo (MVA-mBN226, n=2), withMVA-BN-ZEBOV/GP-VP40 (MVA-mBN255, n=2), or received Tris-buffered saline(TBS) as negative control (placebo group, n=2). Prior to immunizationand weekly before challenge (Days 0, 27, 41 55, 35 and 67) serum wascollected for analysis by Ebola virus Zaire glycoprotein (GP)-specificneutralizing assay (FIG. 11). Four weeks after the last vaccination,animals were challenged with Ebola virus Zaire (Kikwit strain) byintramuscular administration of approximately 100 pfu.

TABLE 6 Study design: Vaccination Challenge Group Vaccine Dose ScheduleVirus Schedule Survival 1 Negative n/a Day 0 + 56 ZEBOV Kikwit 4 weeks0/2 control approx. post last 100 pfu IM vaccination 2 MVA-BN-multi-filo5 × 10⁸ Day 0 + 28 + 56 0/2 TCID₅₀ 3 MVA-BN-ZEBOV/GP-VP40 5 × 10⁸ Day0 + 28 + 56 2/2 TCID₅₀

Vaccination with MVA-BN-ZEBOV/GP-VP40 resulted in neutralizingantibodies detectable already after the prime vaccination, whileMVA-BN-multi-filo did not induce detectable levels of neutralizingantibodies after prime at day 27 (FIG. 11). Animals vaccinated withMVA-BN-ZEBOV/GP-VP40 had higher neutralizing antibody titers thanMVA-BN-multi-filo vaccinated animals throughout all time points ofanalysis. After ZEBOV challenge MVA-BN-multi-filo succumbed by day 7post challenge whereas MVA-BN-ZEBOV/GP-VP40 vaccinated animals survivedwith no symptoms or a transient fever episode.

Example 7: VLP Formation and Protein Expression of GP and VP40

HeLa cells were infected with the indicated viruses at a MOI of 10.After 2 days of infection, supernatants were harvested and VLPs in thecleared supernatants (SNs) were then pelleted through a 20% sucrosecushion by ultracentrifugation (UC-SN). Cellular lysates were preparedby direct lysis of cells in 1× Laemmli buffer. Cell lysates were diluted1:5 prior to separation by SDS-PAGE for immunoblot analysis. UC-SN wasnot diluted prior to SDS-PAGE. ZEBOV-GP was detected using a monoclonalmouse antibody (clone 6D8) from USAMRIID, and ZEBOV-VP40 was detectedusing a purified rabbit polyclonal antibody from IBT Bioservices.

Expression of GP and VP40 was confirmed in the fresh preparations byimmunoblot. Both proteins were present in cellular lysates and were alsoenriched in the UC-SN (FIG. 12C). Expression of the matrix protein VP40is known to be sufficient for the formation of VLPs and no directinteraction of VP40 and GP protein has been reported. To show that GP isindeed incorporated into VLPs together with VP40 GP wasimmunoprecipitated from the SN of infected cells. For this purpose, HeLacells were infected with MVA-ZEBOV/GP-VP40 and control cells withMVA-ZEBOV/GP and BAC-derived MVA wt. BAC-derived MVA wt has beendescribed previously in Meisinger-Henschel et al (Meisinger-Henschel etal. (2010), J Virol. 84(19):9907-9919). The SNs from infected cells weresubjected to immunoprecipitation (IP) using an anti-GP-specificantibody. Aliquots of SNs were treated with 1% Triton X-100 (TX-100) for30 minutes prior to immunoprecipitation, which was previously shown todisrupt the majority of mature enveloped VLPs of the murine leukemiavirus (Davidoff et al. (2012), Virology 433(2):401-409).

The IP-complexes were then analyzed by immunoblot for the presence of GPand of co-precipitated VP40. For immunoprecipitation (IP), cleared SNswere incubated with anti-ZEBOV-GP (clone 6D8, USAMRIID) antibodytogether with Protein G-Agarose (10 μl) at 4° C. overnight. Immunoblotsof the immunoprecipitates were then incubated with antibodies againstZEBOV-GP (monoclonal antibody 6D8) and ZEBOV-VP40 (from IBT). GP proteinwas immunoprecipitated efficiently from the SN of cells expressing onlyGP (FIG. 12D, top panel) which was independent of the presence of VP40.Importantly, VP40 co-immunoprecipitated with GP from supernatants ofMVA-ZEBOV/GP-VP40 infected cells only in the absence of TX-100 (FIG.12D, bottom panel, lane 2), indicating that GP had indeed beenincorporated into VLPs. Since TX-100 is supposed to disrupt VLPs andsince no direct GP-VP40 interaction exists, no VP40 could beco-precipitated in the TX-100 treated samples (FIG. 12D, bottom panel,lane 4). Thus, it was shown that indeed GP presenting VLP were producedupon infection with recombinant MVA.

Example 8: VLP Formation in 293T/17

To possibly get higher VLP concentrations after infection of cells,293T/17 cells for the preparation of fresh VLPs. Protein contents ofpreparations were analyzed for GP and VP40 by Western Blot.

293T/17 cells in T175 culture dishes were infected with the indicatedviruses at a MOI of 10. Supernatants were collected 24 h post infectionand either directly mixed with 3× loading buffer (crude SN) orconcentrated over a 20% sucrose cushion (UZ-prep). Cellular lysates (CL)were prepared in 1× loading buffer. Proteins were separated according tosize by denaturing SDS-PAGE. Immunoblots were incubated with anti-GPantibody (clone 6D8, 1:2500, USAMRIID) or anti-VP40 antibody(polyclonal, 1:1000, IBT) and were developed using a chemiluminescencesubstrate.

Expression of EBOV glycoprotein (GP) was readily detectable afterinfection of cells with MVA-ZEBOV/GP and MVA-ZEBOV/GP-VP40(MVA-filo-VLP), VP40 after infection of MVA-filo-VLP, both in cellularlysate (CL) and supernatant (SN) from infected cells. Surprisingly,MVA-ZEBOV/GP-infected cells seem to express more GP when compared tocells infected with MVA-filo-VLP, whereas with MVA-filo-VLP more GP wasfound in the SN. This possibly reflects the fact that with MVA-filo-VLP,the co-expression of GP and VP40 enhances GP release (in form of VLP)from infected cells. Both, GP and VP40 proteins were present in theUZ-preps, indicating that GP and VP40 were collected by UZ. Some GP andalso VP40 was still present in SN after UZ, although less when comparedto crude SN, especially true for VP40. Thus, VP40—mainly present in formof VLPs, together with GP—is largely depleted from SN, whereas parts ofthe GP pool (possibly in form of pleomorphic particles) remain in the SNafter UZ.

Transmission electron microscopy (TEM) and immuno-electron microscopyanalysis of VLPs from 293T/17 cells showed that MVA-filo-VLPs producedby the respective MVA recombinant were densely decorated with GP, GPspikes lining the entire surface of a filo-VLP. Additionally,preparations from cells infected with MVA-wt or MVA-ZEBOV/GP wereanalyzed by immuno-EM; no VLPs were detected in these samples.

Example 9: Immunogenicity of Heterologous Prime-Boost Immunization inNHP

Four cynomolgus macaques were vaccinated (s.c.) on Days 0 and 28. Twoanimals received as prime 5×10⁸ TCID₅₀ MVA-mBN226 Day 0 and as boost1×10⁹ TCID₅₀ FPV-mBN368 Day 28. One animal received as prime 1×10⁹TCID₅₀ FPV-mBN368 Day 0 and as boost 5×10⁸ TCID₅₀ MVA-mBN226. Onecontrol animal only received TBS. On Days 0, 7, 28, 35 and 49 PBMC wereisolated. Blood was collected at Day 0, 28 and 49 for hematology,clinical chemistry, coagulation parameters, isolation of PBMCs or serumfor analysis of T cell and antibody responses, respectively, and forviral load analysis. Serum samples were analyzed for Ebola specifichumoral responses by ELISA and FRNT. GP- and Vaccinia-specific T cellswere analyzed by in vitro re-stimulation of PBMC with a ZEBOV/GP peptidelibrary with Vaccinia Wyeth, followed by detection of IFN-γ secretingcells by ELISPOT. All animals received an intramuscular (i.m.) challenge(100 pfu) of EBOV Kikwit-9510621 on Day 56. All three animals whoreceived heterologous prime boost with MVA and FPV survived. Fullseroconversion was already obtained after prime which was furtherimproved by the boost.

Description of the Sequence Listing

-   SEQ ID NO:1 [DNA sequence encoding GP-SEBOV-Maleo (GenBank Accession    No. U23069.1)]-   SEQ ID NO:2 [amino acid sequence of GP-SEBOV-Maleo (GenBank    Accession No. U23069.1)]-   SEQ ID NO:3 [DNA sequence encoding NP-SEBOV-Boniface (GenBank    Accession No. AF173836.1)]-   SEQ ID NO:4 [amino acid sequence of NP-SEBOV-Boniface (GenBank    Accession No. AF173836)]-   SEQ ID NO:5 [codon-optimized DNA sequence encoding GP-MARV-Musoke    (GenBank Accession No. ABA87127.1 for protein sequence)]-   SEQ ID NO:6 [amino acid sequence of GP-MARV-Musoke (GenBank    Accession No. ABA87127.1)]-   SEQ ID NO:7 [DNA sequence encoding TTC]-   SEQ ID NO:8 [amino acid sequence of TTC]-   SEQ ID NO:9 [DNA sequence encoding hCD40L]-   SEQ ID NO:10 [amino acid sequence of hCD40L]-   SEQ ID NO:11 [DNA sequence encoding hIL15R-Sushi]-   SEQ ID NO:12 [amino acid sequence of hIL15R-Sushi]-   SEQ ID NO:13 [DNA sequence encoding human LFA-3/CD58 (EMBL-CDS    Accession No. CAA75083.1)]-   SEQ ID NO:14 [amino acid sequence of human LFA-3/CD58    (UniProtKB/SwissProt Accession No. P19256)]-   SEQ ID NO:15 [DNA sequence encoding human ICAM-1/CD54 (GenBank    Accession No. BT006854)]-   SEQ ID NO:16 [amino acid sequence of human ICAM-1/CD54    (UniProtKB/SwissProt Accession No. P05362)]-   SEQ ID NO:17 [DNA sequence encoding human B7.1/CD80 (EMBL-CDS    Accession No. AAA58390.1)]-   SEQ ID NO:18 [amino acid sequence of human B7.1/CD80    (UniProtKB/SwissProt Accession No. P33681)]-   SEQ ID NO:19 [codon-optimized DNA encoding GP-ZEBOV-Mayinga (GenBank    Accession No. ABX75367.1)]-   SEQ ID NO:20 [amino acid sequence of GP-ZEBOV-Mayinga (GenBank    Accession No. ABX75367.1)]-   SEQ ID NO:21 [DNA sequence encoding W B5R anchor]-   SEQ ID NO:22 [amino acid sequence of W B5R anchor]-   SEQ ID NO:23 [DNA sequence of PrS promoter]-   SEQ ID NO:24 [DNA sequence of PrS5E promoter: 1× (PrS)+5× (Pr7.5e)]-   SEQ ID NO:25 [DNA sequence of Pr7.5 promoter]-   SEQ ID NO:26 [DNA sequence of the FPV-40K promoter of FPV-   SEQ ID NO:27 [DNA sequence of PrLE1 promoter—1× (ATI)+5× (Pr7.5e)]-   SEQ ID NO:28 [codon-optimized DNA sequence encoding NP-EBOV-Cdl    (GenBank Accession No. ACI28629.1)]-   SEQ ID NO:29 [amino acid sequence of NP-EBOV-Cdl (GenBank Accession    No. ACI28629.1)]-   SEQ ID NO:30 [codon optimized DNA sequence encoding GP-SEBOV-Gulu    (GenBank Accession No. AAU43887.1)]-   SEQ ID NO:31 [amino acid sequence of GP-SEBOV-Gulu (GenBank    Accession No. AAU43887.1)]-   SEQ ID NO:32 [DNA sequence of PrLE1 promoter—1× (ATI)+4× (Pr7.5e)]-   SEQ ID NO:33 [codon optimized DNA sequence DNA sequence encoding    VP40-ZEBOV-Mayinga sequence]-   SEQ ID NO:34 [amino acid sequence of VP40-ZEBOV-Mayinga sequence]-   SEQ ID NO:35 [Pr13.5 promoter sequence]-   SEQ ID NO:36 [codon optimized DNA sequence DNA sequence encoding    GP-MARV-Angola]-   SEQ ID NO:37 [amino acid sequence of GP-MARV-Angola]

The invention claimed is:
 1. A recombinant MVA vector comprising a firstnucleic acid encoding at least one immunogenic protein of a MARVenvelope glycoprotein (GP); a second nucleic acid encoding animmunogenic protein of Zaire Ebola virus (ZEBOV) envelope glycoprotein;a third nucleic acid encoding an immunogenic protein of Sudan Ebolavirus (SEBOV) envelope glycoprotein; and a fourth nucleic acid encodingan immunogenic protein of Ebola virus Ivory Coast nucleoprotein.
 2. Therecombinant MVA vector of claim 1, wherein the MARV envelopeglycoprotein is full-length MARV-Musoke envelope glycoprotein.
 3. Therecombinant MVA vector of claim 1, wherein the first nucleic acidencodes an immunogenic protein comprising the sequence set forth in SEQID NO:6.
 4. The recombinant MVA vector of claim 3, wherein the firstnucleic acid comprises the sequence set forth in SEQ ID NO:5.
 5. Therecombinant MVA vector of claim 1 that comprises a nucleic acid encodingan immunogenic protein having a sequence selected from the groupconsisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQID NO:29, SEQ ID NO:31, and SEQ ID NO:37.
 6. The recombinant MVA vectorof claim 1 that comprises a nucleic acid encoding an immunogenic proteincomprising the sequence set forth in SEQ ID NO:6, SEQ ID NO:20, SEQ IDNO:29, or SEQ ID NO:31.
 7. The recombinant MVA vector of claim 6,wherein said nucleic acid comprises the sequence set forth in SEQ IDNO:5, SEQ ID NO:19, SEQ ID NO:28, or SEQ ID NO:30.
 8. The recombinantMVA vector of claim 1, wherein the administration provides protectiveimmunity or a protective immune response in the subject.
 9. Therecombinant MVA vector of claim 1, wherein the recombinant MVA vectorcomprises at least one nucleic acid encoding the sequences set forth inSEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:29, and SEQ ID NO:31.
 10. Therecombinant MVA vector of claim 1 further comprising a nucleic acidencoding CD40L.
 11. The recombinant MVA vector of claim 10, wherein theCD40L comprises the amino acid sequence set forth in SEQ ID NO:10. 12.The recombinant MVA vector of claim 11, wherein the nucleic acidencoding CD40L comprises the sequence set forth in SEQ ID NO:9.